Production of sialylated n-glycans in lower eukaryotes

ABSTRACT

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and/or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/554,126, filed Jul. 20, 2012, now pending, which is a continuation ofU.S. application Ser. No. 12/819,305, filed Jun. 21, 2010, now U.S. Pat.No. 8,268,609, issued Sep. 18, 2012, which is a continuation of U.S.application Ser. No. 11/429,672, filed May 5, 2006, now U.S. Pat. No.7,863,020, issued Jan. 4, 2011, which is a continuation-in-part of U.S.application Ser. No. 11/108,088, filed Apr. 15, 2005, now U.S. Pat. No.7,795,002, issued Sep. 14, 2010, which is a continuation-in-part of U.S.application Ser. No. 11/084,624, filed Mar. 17, 2005, now abandoned,which claims the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication No. 60/554,139, filed Mar. 17, 2004, now expired, andcontinuation-in-part of U.S. application Ser. No. 10/371,877, filed Feb.20, 2003, now U.S. Pat. No. 7,449,308, issued Nov. 11, 2008.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name GFI BIO 0025 US CNT 5 SEQLIST.TXT-”, creation date of Mar. 12,2015, and a size of 105 KB. This sequence listing submitted via EFS-Webis part of the specification and is herein incorporated by reference inits entirety.—

FIELD OF THE INVENTION

The present invention is directed to methods and compositions by whichnon-human eukaryotic host cells, such as fungi or other eukaryoticcells, can be genetically modified to produce glycosylated proteins(glycoproteins) having patterns of glycosylation similar to those ofglycoproteins produced by animal cells, especially human cells, whichare useful as human or animal therapeutic agents. In particular thisapplication relates to methods and compositions for the production ofsialylated glycoproteins in non-human eukaryotic host cells that do notnormally produce sialylated glycoproteins.

BACKGROUND OF THE INVENTION Glycosylation Pathways in Humans and LowerEukaryotes

After DNA is transcribed and translated into a protein, furtherpost-translational processing involves the attachment of sugar residues,a process known as glycosylation. Different organisms produce differentglycosylation enzymes (glycosyltransferases and glycosidases), and havedifferent substrates (nucleotide sugars) available, so that theglycosylation patterns as well as composition of the individualoligosaccharides, even of the same protein, will be different dependingon the host system in which the particular protein is being expressed.Bacteria typically do not glycosylate proteins, and if so only in a veryunspecific manner (Moens, 1997). (Throughout the specification,scientific publications will be cited by reference to the senior authorand publication year; complete citations are found at the end of thespecification.) Lower eukaryotes such as filamentous fungi and yeast addprimarily mannose and mannosylphosphate sugars. The resulting glycan isknown as a “high-mannose” type glycan or a mannan. Plant cells andinsect cells (such as Sf9 cells) glycosylate proteins in yet anotherway. By contrast, in higher eukaryotes such as humans, the nascentoligosaccharide side chain may be trimmed to remove several mannoseresidues and elongated with additional sugar residues that typically arenot found in the N-glycans of lower eukaryotes. See, e.g., Bretthauer,1999; Martinet, 1998; Weikert, 1999; Malissard, 2000; Jarvis 1998; andTakeuchi, 1997.

Synthesis of a mammalian-type oligosaccharide structure begins with aset of sequential reactions in the course of which sugar residues areadded and removed while the protein moves along the secretory pathway inthe host organism. The enzymes which reside along the glycosylationpathway of the host organism or cell determine the resultingglycosylation patterns of secreted proteins. Thus, the resultingglycosylation pattern of proteins expressed in lower eukaryotic hostcells differs substantially from the glycosylation pattern of proteinsexpressed in higher eukaryotes such as humans and other mammals(Bretthauer, 1999). The structure of a typical fungal N-glycan is shownin FIG. 1A.

The early steps of human glycosylation can be divided into at least twodifferent phases: (i) lipid-linked Glc₃Man₉GlcNAc₂ oligosaccharides areassembled by a sequential set of reactions at the membrane of theendoplasmic reticulum (ER) and (ii) the transfer of this oligosaccharidefrom the lipid anchor dolichyl pyrophosphate onto de novo synthesizedprotein. The site of the specific transfer is defined by an asparagine(Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaa can be any aminoacid except proline (Gavel, 1990). Further processing by glucosidasesand mannosidases occurs in the ER before the nascent glycoprotein istransferred to the early Golgi apparatus, where additional mannoseresidues are removed by Golgi specific alpha (α)-1,2-mannosidases.Processing continues as the protein proceeds through the Golgi. In themedial Golgi, a number of modifying enzymes, includingN-acetylglucosaminyl Transferases (GnTI, GnTII, GnTIII, GnTIV and GnTV),mannosidase II and fucosyltransferases, add and remove specific sugarresidues. Finally, in the trans-Golgi, galactosyltranferases (GalT) andsialyltransferases (ST) produce a glycoprotein structure that isreleased from the Golgi. It is this structure, characterized by bi-,tri- and tetra-antennary structures, containing galactose, fucose,N-acetylglucosamine and a high degree of terminal sialic acid, thatgives glycoproteins their human characteristics. The structure of atypical human N-glycan is shown in FIG. 1B.

In nearly all eukaryotes, glycoproteins are derived from a commonlipid-linked oligosaccharide precursorGlc₃Man₉GlcNAc₂-dolichol-pyrophosphate. Within the endoplasmicreticulum, synthesis and processing of dolichol pyrophosphate boundoligosaccharides are identical between all known eukaryotes. However,further processing of the core oligosaccharide by fungal cells, e.g.,yeast, once it has been transferred to a peptide leaving the ER andentering the Golgi, differs significantly from humans as it moves alongthe secretory pathway and involves the addition of several mannosesugars.

In yeast, these steps are catalyzed by Golgi residingmannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentiallyadd mannose sugars to the core oligosaccharide. The resulting structureis undesirable for the production of human-like proteins and it is thusdesirable to reduce or eliminate mannosyltransferase activity. Mutantsof Saccharomyces cerevisiae (S. cerevisiae), deficient inmannosyltransferase activity (for example och1 or mnn9 mutants) havebeen shown to be non-lethal and display reduced mannose content in theoligosaccharide of yeast glycoproteins. Other oligosaccharide processingenzymes, such as mannosylphosphate transferase, may also have to beeliminated depending on the host's particular endogenous glycosylationpattern.

Sugar Nucleotide Precursors

The N-glycans of animal glycoproteins typically include galactose,fucose, and terminal sialic acid. These sugars are not found onglycoproteins produced in yeast and filamentous fungi. In humans andother non-human eukaryotic cells, the full range of sugar nucleotideprecursors (e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) aresynthesized in the cytosol and transported into the Golgi, where theyare attached to the core oligosaccharide by glycosyltransferases.(Sommers, 1981; Sommers, 1982; Perez, 1987).

Glycosyl transfer reactions typically yield a side product which is anucleoside diphosphate or monophosphate. While monophosphates can bedirectly exported in exchange for nucleoside triphosphate sugars by anantiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleavedby phosphatases (e.g. GDPase) to yield nucleoside monophosphates andinorganic phosphate prior to being exported. This reaction is importantfor efficient glycosylation; for example, GDPase from Saccharomycescerevisiae (S. cerevisiae) has been found to be necessary formannosylation. However that GDPase has 90% reduced activity toward UDP(Berninsone, 1994). Lower eukaryotes typically lack UDP-specificdiphosphatase activity in the Golgi since they do not utilize UDP-sugarprecursors for Golgi-based glycoprotein synthesis. Schizosaccharomycespombe, a yeast found to add galactose residues to cell wallpolysaccharides (from UDP-galactose) has been found to have specificUDPase activity, indicating the potential requirement for such an enzyme(Berninsone, 1994). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productmay be important to prevent glycosyl-transferase inhibition in the lumenof the Golgi (Khatara, 1974). See Berninsone, 1995; Beaudet, 1998.

Sequential Processing of N-Glycans by Compartmentalized EnzymeActivities

Sugar transferases and glycosidases (e.g., mannosidases) line the inner(luminal) surface of the ER and Golgi apparatus and thereby provide a“catalytic” surface that allows for the sequential processing ofglycoproteins as they proceed through the ER and Golgi network. Themultiple compartments of the cis, medial, and trans Golgi and thetrans-Golgi Network (TGN), provide the different localities in which theordered sequence of glycosylation reactions can take place. As aglycoprotein proceeds from synthesis in the ER to full maturation in thelate Golgi or TGN, it is sequentially exposed to different glycosidases,mannosidases and glycosyltransferases such that a specific carbohydratestructure may be synthesized. Much work has been dedicated to revealingthe exact mechanism by which these enzymes are retained and anchored totheir respective organelle. The evolving picture is complex but evidencesuggests that stem region, membrane spanning region and cytoplasmictail, individually or in concert, direct enzymes to the membrane ofindividual organelles and thereby localize the associated catalyticdomain to that locus (see, e.g., Gleeson, 1998).

In some cases, these specific interactions were found to function acrossspecies. For example, the membrane spanning domain of α2,6-ST from rats,an enzyme known to localize in the trans-Golgi of the animal, was shownto also localize a reporter gene (invertase) in the yeast Golgi(Schwientek, 1995). However, the very same membrane spanning domain aspart of a full-length α2,6-ST was retained in the ER and not furthertransported to the Golgi of yeast (Krezdorn, 1994). A full length GalTfrom humans was not even synthesized in yeast, despite demonstrably hightranscription levels. In contrast, the transmembrane region of the samehuman GalT fused to an invertase reporter was able to directlocalization to the yeast Golgi, albeit it at low production levels.Schwientek and co-workers have shown that fusing 28 amino acids of ayeast mannosyltransferase (MNT1), a region containing a cytoplasmictail, a transmembrane region and eight amino acids of the stem region,to the catalytic domain of human GalT are sufficient for Golgilocalization of an active GalT. Other galactosyltransferases appear torely on interactions with enzymes resident in particular organellesbecause, after removal of their transmembrane region, they are stillable to localize properly.

Improper localization of a glycosylation enzyme may prevent properfunctioning of the enzyme in the pathway. For example, Aspergillusnidulans, which has numerous α-1,2-mannosidases (Eades, 2000), does notadd GlcNAc to Man₅GlcNAc₂ when transformed with the rabbit GnTI gene,despite a high overall level of GnTI activity (Kalsner et al., 1995).GnTI, although actively expressed, may be incorrectly localized suchthat the enzyme is not in contact with both of its substrates:UDP-GlcNAc and a productive Man₅GlcNAc₂ substrate (not all Man₅GlcNAc₂structures are productive; see below). Alternatively, the host organismmay not provide an adequate level of UDP-GlcNAc in the Golgi or theenzyme may be properly localized but nevertheless inactive in its newenvironment. In addition, Man₅GlcNAc₂ structures present in the hostcell may differ in structure from Man₅GlcNAc₂ found in mammals. Marasand coworkers found that about one third of the N-glycans fromcellobiohydrolase I (CBHI) obtained from T. reesei can be trimmed toMan₅GlcNAc₂ by A. saitoi 1,2 mannosidase in vitro. Fewer than 1% ofthose N-glycans, however, could serve as a productive substrate forGnTI. The mere presence of Man₅GlcNAc₂, therefore, does not assure thatfurther processing to Man₅GlcNAc₂ can be achieved. It is formation of aproductive, GnTI-reactive Man₅GlcNAc₂ structure that is required.Although Man₅GlcNAc₂ could be produced in the cell (about 27 mol %),only a small fraction could be converted to Man₅GlcNAc₂ (less than about5%, see WO 01/14522).

To date, there is no reliable way of predicting whether a particularheterologously expressed glycosyltransferase or mannosidase in a lowereukaryote will be (1), sufficiently translated (2), catalytically activeor (3) located to the proper organelle within the secretory pathway.Because all three of these are necessary to affect glycosylationpatterns in lower eukaryotes, a systematic scheme to achieve the desiredcatalytic function and proper retention of enzymes in the absence ofpredictive tools, which are currently not available, would be desirable.

Production of Therapeutic Glycoproteins

A significant number of proteins isolated from humans or animals arepost-translationally modified, with glycosylation being one of the mostsignificant modifications. An estimated 70% of all therapeutic proteinsare glycosylated and thus currently rely on a production system (i.e.,host cell) that is able to glycosylate in a manner similar to humans.Several studies have shown that glycosylation plays an important role indetermining the (1) immunogenicity, (2) pharmacokinetic properties, (3)trafficking, and (4) efficacy of therapeutic proteins. It is thus notsurprising that substantial efforts by the pharmaceutical industry havebeen directed at developing processes to obtain glycoproteins that areas “humanoid” or “human-like” as possible. To date, most glycoproteinsare made in a mammalian host system. This may involve the geneticengineering of such mammalian cells to enhance the degree of sialylation(i.e., terminal addition of sialic acid) of proteins expressed by thecells, which is known to improve pharmacokinetic properties of suchproteins. Alternatively, one may improve the degree of sialylation by invitro addition of such sugars using known glycosyltransferases and theirrespective nucleotide sugars (e.g., 2,3-sialyltransferase and CMP-sialicacid).

While most higher eukaryotes carry out glycosylation reactions that aresimilar to those found in humans, recombinant human proteins expressedin the above mentioned host systems invariably differ from their“natural” human counterpart (Raju, 2000). Extensive development work hasthus been directed at finding ways to improve the “human character” ofproteins made in these expression systems. This includes theoptimization of fermentation conditions and the genetic modification ofprotein expression hosts by introducing genes encoding enzymes involvedin the formation of human-like glycoforms (Werner, 1998; Weikert, 1999;Andersen, 1994; Yang, 2000) Inherent problems associated with allmammalian expression systems have not been solved.

Most, if not all, currently produced therapeutic glycoproteins aretherefore expressed in mammalian cells and much effort has been directedat improving (i.e., “humanizing”) the glycosylation pattern of theserecombinant proteins. Changes in medium composition as well as theco-expression of genes encoding enzymes involved in human glycosylationhave been successfully employed (see, for example, Weikert, 1999).

Glycoprotein Production Using Eukaryotic Microorganisms

The lack of a suitable mammalian expression system is a significantobstacle to the low-cost and safe production of recombinant humanglycoproteins for therapeutic applications. It would be desirable toproduce recombinant proteins similar to their mammalian, e.g., human,counterparts in lower eukaryotes (fungi and yeast). Production ofglycoproteins via the fermentation of microorganisms would offernumerous advantages over existing systems. Although the coreoligosaccharide structure transferred to a protein in the endoplasmicreticulum is basically identical in mammals and lower eukaryotes,substantial differences have been found in the subsequent processingreactions which occur in the Golgi apparatus of fungi and mammals. Infact, even amongst different lower eukaryotes there exist a greatvariety of glycosylation structures. This has historically prevented theuse of lower eukaryotes as hosts for the production of recombinant humanglycoproteins despite otherwise notable advantages over mammalianexpression systems.

Therapeutic glycoproteins produced in a microorganism host such as yeastutilizing the endogenous host glycosylation pathway differ structurallyfrom those produced in mammalian cells and typically show greatlyreduced therapeutic efficacy. Such glycoproteins are typicallyimmunogenic in humans and show a reduced half-life (and thusbioactivity) in vivo after administration (Takeuchi, 1997). Specificreceptors in humans and animals (i.e., macrophage mannose receptors) canrecognize terminal mannose residues and promote the rapid clearance ofthe foreign glycoprotein from the bloodstream. Additional adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity.

Yeast and filamentous fungi have both been successfully used for theproduction of recombinant proteins, both intracellular and secreted(Cereghino, 2000; Harkki, 1989; Berka, 1992; Svetina, 2000). Variousyeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, andHansenula polymorphs, have played particularly important roles aseukaryotic expression systems because they are able to grow to high celldensities and secrete large quantities of recombinant protein. Likewise,filamentous fungi, such as Aspergillus niger, Fusarium sp., Neurosporacrassa and others, have been used to efficiently produce glycoproteinsat the industrial scale. However, as noted above, glycoproteinsexpressed in any of these eukaryotic microorganisms differ substantiallyin N-glycan structure from those in animals. This has prevented the useof yeast or filamentous fungi as hosts for the production of manytherapeutic glycoproteins.

Although glycosylation in yeast and fungi is very different than inhumans, some common elements are shared. The first step, the transfer ofthe core oligosaccharide structure to the nascent protein, is highlyconserved in all eukaryotes including yeast, fungi, plants and humans(compare FIGS. 1A and 1B). Subsequent processing of the coreoligosaccharide, however, differs significantly in yeast and involvesthe addition of several mannose sugars. This step is catalyzed bymannosyltransferases residing in the Golgi (e.g. OCH1, MNT1, MNN1,etc.), which sequentially add mannose sugars to the coreoligosaccharide. The resulting structure is undesirable for theproduction of humanoid proteins and it is thus desirable to reduce oreliminate mannosyltransferase activity. Mutants of S. cerevisiaedeficient in mannosyltransferase activity (e.g. och1 or mnn9 mutants)have shown to be non-lethal and display a reduced mannose content in theoligosaccharide of yeast glycoproteins. Other oligosaccharide processingenzymes, such as mannosylphosphate transferase, may also have to beeliminated depending on the host's particular endogenous glycosylationpattern. After reducing undesired endogenous glycosylation reactions,the formation of complex N-glycans has to be engineered into the hostsystem. This requires the stable expression of several enzymes andsugar-nucleotide transporters. Moreover, one has to localize theseenzymes so that a sequential processing of the maturing glycosylationstructure is ensured.

Several efforts have been made to modify the glycosylation pathways ofeukaryotic microorganisms to provide glycoproteins more suitable for useas mammalian therapeutic agents. However, N-glycans resembling thosemade in human cells (e.g., with complex or hybrid glycan structures)were not obtained.

Yeasts produce a variety of mannosyltransferases (e.g.,1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham, 1991,1,2-mannosyltransferases (e.g. KTR/KRE family from S. cerevisiae),1,6-mannosyltransferases (e.g., OCH1 from S. cerevisiae),mannosylphosphate transferases and their regulators (e.g., MNN4 and MNN6from S. cerevisiae) and additional enzymes that are involved inendogenous glycosylation reactions. Many of these genes have beendeleted individually giving rise to viable organisms having alteredglycosylation profiles.

While it is useful to engineer strains that are able to produceMan₅GlcNAc₂ as the primary N-glycan structure, any attempt to furthermodify these high mannose precursor structures to more closely resemblehuman glycans requires additional in vivo or in vitro steps. Methods tofurther humanize glycans from fungal and yeast sources in vitro aredescribed in U.S. Pat. No. 5,834,251. As discussed above, however, ifMan₅GlcNAc₂ is to be further humanized in vivo, one has to ensure thatthe generated Man₅GlcNAc₂ structures are, in fact, generatedintracellularly and not the product of mannosidase activity in themedium. Complex N-glycan formation in yeast or fungi will require highlevels of Man₅GlcNAc₂ to be generated within the cell because onlyintracellular Man₅GlcNAc₂ glycans can be further processed to hybrid andcomplex N-glycans in vivo. In addition, one has to demonstrate that themajority of Man₅GlcNAc₂ structures generated are in fact a substrate forGnTI and thus allow the formation of hybrid and complex N-glycans.

Accordingly, the need exists for methods to produce glycoproteinscharacterized by a high intracellular Man₅GlcNAc₂ content which can befurther processed into human-like glycoprotein structures in non-humaneukaryotic host cells, and particularly in yeast and filamentous fungi.

Addition of Sialic Acid to N-Glycans

Sialic acids (Sia) are a unique group of N- or O-substituted derivativesof N-acetylneuraminic acid (Neu5Ac) which are ubiquitous in animals ofthe deuterostome lineage, from starfish to humans. In other organisms,including most plants, protists, Archaea, and eubacteria, thesecompounds are thought to be absent (Warren, 1994). Exceptions have beenidentified, all of which are in pathogenic organisms, including certainbacteria, protozoa and fungi (Kelm, 1997; Parodi, 1993; Alviano, 1999).The mechanism by which pathogenic fungi, including Cryptococcusneoformans and Candida albicans, acquire sialic acid on cell surfaceglycoproteins and glycolipids remains undetermined (Alviano, 1999). Whenthese organisms are grown in sialic acid-free media, sialic acidresidues are found on cellular glycans, suggesting de novo synthesis ofsialic acid. To date, no enzymes have been identified in fungi that areinvolved in the biosynthesis of sialic acid. The mechanism by whichprotozoa sialylate cell surface glycans has been well-characterized.Protozoa, such as Trypanosoma cruzi, possess an external trans-sialidasethat adds sialic acid to cell surface glycoproteins and glycolipids in aCMP-Sia independent mechanism (Parodi, 1993). The identification of asimilar trans-sialidase in fungi would help to elucidate the mechanismof sialic acid transfer on cellular glycans, but such a protein has notyet been identified or isolated.

Despite the absence and/or ambiguity of sialic acid biosynthesis infungi, sialic acid biosynthesis in pathogenic bacteria and mammaliancells is well understood. A group of pathogenic bacteria have beenidentified that synthesize sialic acids de novo to generate sialylatedglycolipids that occur on the cell surface (Vimr, 1995). Although sialicacids on the surface of these pathogenic organisms are predominantlythought to be a means of evading the host immune system, these samesialic acid molecules are also involved in many processes in higherorganisms, including protein targeting, cell-cell interaction,cell-substrate recognition and adhesion (Schauer, 2000).

The presence of sialic acids can affect biological activity andhalf-life of glycoproteins in vivo (MacDougall, 1999). For example, theimportance of sialic acids has been demonstrated in studies of humanerythropoietin (hEPO). The terminal sialic acid residues on thecarbohydrate chains of the N-linked glycan of this glycoprotein preventrapid clearance of hEPO from the blood and improve in vivo activity.Asialylated-hEPO (asialo-hEPO), which terminates in a galactose residue,has dramatically decreased erythropoietic activity in vivo. Thisdecrease is caused by the increased clearance of the asialo-hEPO by thehepatic asialoglycoprotein receptor (Fukuda, 1989; Spivak, 1989; Spivak,1989). Similarly, the absence of terminal sialic acid on manytherapeutic glycoproteins can reduce efficacy in vivo, and thus requiremore frequent patient dosing regimes.

SUMMARY OF THE INVENTION

Host cells and cell lines having genetically modified glycosylationpathways that allow them to carry out a specified sequence of enzymaticreactions which mimic the processing of glycoproteins in mammals,especially in humans, have been developed. Recombinant proteinsexpressed in these engineered hosts yield glycoproteins more similar, ifnot substantially identical, to their mammalian, e.g., humancounterparts. Moreover, substantially homogeneous glycoproteinpopulations having particular desired glycan structures may be produced.Host cells of the invention, e.g., lower eukaryotic micro-organisms andother non-human, eukaryotic host cells grown in culture, are modified toproduce N-glycans produced along human glycosylation pathways. This isachieved using a combination of engineering and/or selection of strainsthat: (1) do not express certain enzymes which create the undesirablestructures characteristic of the fungal glycoproteins; (2) expressheterologous enzymes selected either to have optimal activity under theconditions present in the host cell where activity is to be achieved; or(3) combinations thereof; wherein the genetically engineered host cellexpresses at least one heterologous enzyme activity required to producea “human-like” glycoprotein. Host cells of the invention may be modifiedfurther by heterologous expression of one or more activities such asglycosyltransferases, glycosidase (such as mannosidases), sugarnucleotide transporters, and the like, to become strains for theproduction of mammalian, e.g., human therapeutic glycoproteins.

The present invention thus provides a glycoprotein production methodusing (1) a lower eukaryotic host such as a unicellular or filamentousfungus, or (2) any eukaryotic organism, e.g., a non-human eukaryoticcell or organism that has a different glycosylation pattern from humans,to modify the glycosylation composition and structures of the proteinsmade in a host organism (“host cell”) so that they resemble more closelycarbohydrate structures found in mammalian, e.g., human proteins. Theprocess allows one to obtain an engineered host cell in which desirablegene(s), e.g., one(s) involved in glycosylation, is(are) expressed andits (their) product(s) targeted to a subcellular location in the hostcell by methods that are well-established in the scientific literatureand generally known to the artisans in the field of protein expression.For the production of therapeutic proteins, this method may be adaptedto engineer cell lines in which any desired glycosylation structure maybe obtained on proteins expressed in the engineered cells.

In one embodiment, N-glycans made in the host cells have a Man₅GlcNAc₂core structure which may then be modified further by heterologousexpression of one or more enzymes, e.g., glycosyltransferases,glycosidases such as mannosidases, sugar transporters and the like, toyield modified glycoproteins, e.g., having human-like complex or hybridN-glycan structures. In certain embodiments, N-glycans made in the hostcells have a complex Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ structure which maythen be modified further by heterologous expression of one or moreenzymes, e.g., glycosyltransferases, glycosidases such as mannosidases,sugar transporters and the like, to yield modified glycoproteins, e.g.,having human-like complex or hybrid N-glycan structures. In otherembodiments, N-glycans made in the host cells have a hybridGalGlcNAcMan₅GlcNAc₂ structure which may then be modified further byheterologous expression of one or more enzymes, e.g.,glycosyltransferases, glycosidases such as mannosidases, sugartransporters and the like, to yield modified glycoproteins, e.g., havinghuman-like complex or hybrid N-glycan structures.

In one embodiment, glycoproteins made in the engineered host cells havea complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform of N-glycan.In other embodiments, glycoproteins made in the engineered host cellshave a hybrid NANAGalGlcNAcMan₅GlcNA₂ glycoform of N-glycan. Thus, inone embodiment, glycoprotein compositions of the present invention maycomprise predominantly complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂glycoforms. In other embodiments, glycoprotein compositions of thepresent invention will comprise predominantly hybridNANAGalGlcNAcMan₅GlcNA₂ glycoforms. Methods for producing theMan₅GlcNAc₂ core structure and its modification to form either complexand hybrid glycoforms are provided in Gerngross, WO02/00879 and U.S.Pat. No. 7,029,872, the specification of which are hereby incorporatedherein by reference for its disclosure cited herein.

Accordingly, in one embodiment, the invention provides a method forproducing a human-like glycoprotein in a non-human eukaryotic host cell.The host cell of the invention is selected or engineered to be depletedin 1,6-mannosyltransferase activities which would otherwise add mannoseresidues onto the N-glycan on a glycoprotein. One or more enzymes(enzymatic activities) are introduced into the host cell which enablethe production of a Man₅GlcNAc₂ carbohydrate structure at a high yield,e.g., at least 30 mole percent. In a preferred embodiment, at least 10%of the Man₅GlcNAc₂ produced within the host cell is a productivesubstrate for GnTI and thus for further glycosylation reactions in vivoand/or in vitro that produce a finished N-glycan that is similar oridentical to that formed in mammals, especially humans.

In another embodiment, a nucleic acid molecule encoding one or moreenzymes for production of a Man₅GlcNAc₂ carbohydrate structure isintroduced into a host cell selected or engineered to be depleted in1,6-mannosyltransferase activities. In one preferred embodiment, atleast one enzyme introduced into the host cell is selected to haveoptimal activity at the pH of the subcellular location where thecarbohydrate structure is produced. In another preferred embodiment, atleast one enzyme is targeted to a host subcellular organelle where theenzyme will have optimal activity, e.g., by means of a chimeric proteincomprising a cellular targeting signal peptide not normally associatedwith the enzyme.

The invention further provides isolated nucleic acid molecules andvectors comprising such molecules which encode an initiatingα1,6-mannosyltransferase activity isolated from P. pastoris or from K.lactis. These nucleic acid molecules comprise sequences that arehomologous to the OCH1 gene in S. cerevisiae. These and homologoussequences are useful for constructing host cells which will nothypermannosylate the N-glycan of a glycoprotein.

In another embodiment, the host cell is engineered to express aheterologous glycosidase, e.g., by introducing into the host one or morenucleic acid molecules encoding the glycosidase. In one embodiment, anucleic acid molecule encodes one or more mannosidase activitiesinvolved in the production of Man₅GlcNAc₂ from Man₈GlcNAc₂ orMan₉GlcNAc₂. In a preferred embodiment, at least one of the encodedmannosidase activities has a pH optimum within 1.4 pH units of theaverage pH optimum of other representative enzymes in the organelle inwhich the mannosidase activity is localized, or has optimal activity ata pH of between about 5.1 and about 8.0, preferably between about 5.5and about 7.5. Preferably, the heterologous enzyme is targeted to theendoplasmic reticulum, the Golgi apparatus or the transport vesiclesbetween ER and Golgi of the host organism, where it trims N-glycans suchas Man₈GlcNAc₂ to yield high levels of Man₅GlcNAc₂.

In another embodiment, the host cell is engineered to express aheterologous galactosyltransferase. In yet another embodiment, the hostcell is engineered to express a heterologous sialyltransferase or atrans-sialidase.

In certain embodiments, the glycosylation enzyme is targeted to asubcellular location by forming a fusion protein between a catalyticdomain of the enzyme and a cellular targeting signal peptide, e.g., bythe in-frame ligation of a DNA fragment encoding a cellular targetingsignal peptide with a DNA fragment encoding a glycosylation enzyme orcatalytically active fragment thereof.

In certain embodiments, the glycosylation pathway of a host is modifiedto express a sugar nucleotide transporter. In a preferred embodiment, anucleotide diphosphatase enzyme is also expressed. The transporter anddiphosphatase improve the efficiency of engineered glycosylation steps,by providing the appropriate substrates for the glycosylation enzymes inthe appropriate compartments, reducing competitive product inhibition,and promoting the removal of nucleoside diphosphates.

In another embodiment, the host cell is engineered to express afunctional CMP-sialic acid (CMP-Sia) biosynthetic pathway.

In another embodiment, a method of engineering a CMP-Sia biosyntheticpathway into a non-human eukaryotic cell is provided. The methodinvolves the cloning and expression of several enzymes of mammalianorigin, bacterial origin or both, in a host cell, particularly a fungalhost cell or other host cell that lacks endogenous sialylation or thatcan benefit from increased levels of CMP-Sia. The engineered CMP-Siabiosynthetic pathway is useful for producing sialylated glycolipids,O-glycans and N-glycans in vivo. The present invention is thus usefulfor facilitating the generation of sialylated therapeutic glycoproteinsin non-human host cells lacking endogenous sialylation or in non-humanhost cells lacking adequate levels of endogenous sialylation. Examplesof non-human host cells lacking adequate levels of endogenoussialylation include lower eukaryotic host cells, insect cells and plantcells. Thus, in certain embodiments of the invention, the host cells areengineered to produce sialylated glycoproteins where none wouldotherwise be produced endogenously. In other embodiments, of theinvention, the host cells are engineered to increase the level ofsialylated glycoproteins above native endogenous levels.

The present invention also provides a combinatorial nucleic acid libraryuseful for making fusion constructs which can target a desired proteinor polypeptide fragment, e.g., an enzyme involved in glycosylation or acatalytic domain thereof, to a selected subcellular region of a hostcell. In one preferred embodiment, the combinatorial nucleic acidlibrary comprises (a) nucleic acid sequences encoding different cellulartargeting signal peptides and (b) nucleic acid sequences encodingdifferent polypeptides to be targeted. Nucleic acid sequences of orderived from (a) and (b) are ligated together to produce fusionconstructs, at least one of which encodes a functional protein domain(e.g., a catalytic domain of an enzyme) ligated in-frame to aheterologous cellular targeting signal peptide, i.e., one which itnormally does not associate with.

The invention also provides a method for modifying the glycosylationpathway of a host cell (e.g., any eukaryotic host cell, including ahuman host cell) using enzymes involved in modifying N-glycans includingglycosidases and glycosyltransferases; by transforming the host cellwith a nucleic acid (e.g., a combinatorial) library of the invention toproduce a genetically mixed cell population expressing at least one andpreferably two or more distinct chimeric glycosylation enzymes having acatalytic domain ligated in-frame to a cellular targeting signal peptidewhich it normally does not associate with. A host cell having a desiredglycosylation phenotype may optionally be selected from the population.Host cells modified using the library and associated methods of theinvention are useful, e.g., for producing glycoproteins having aglycosylation pattern similar or identical to those produced in mammals,especially humans.

In another aspect, the combinatorial library of the present inventionenables production of one or a combination of catalytically activeglycosylation enzymes, which successfully localize to intracellularcompartments in which they function efficiently in theglycosylation/secretory pathway. Preferred enzymes convert(α-1,2-Man)₃₋₉ Man₅GlcNAc₂ to Man₅GlcNAc₂ at high efficiency in vivo. Inaddition, the invention provides eukaryotic host strains, and inparticular, yeasts, fungal, insect, plant, plant cells, algae and insectcell hosts, capable of producing glycoprotein intermediates or productswith Man₅GlcNAc₂ and/or GlcNAcMan₅GlcNAc₂ as the predominant N-glycan.

The present invention also provides methods using the combinatoriallibrary for producing, in vivo, glycoprotein intermediates or productswith predominantly Man₅GlcNAc₂ or GlcNAcMan₅GlcNAc₂ N-glycans covalentlyattached to proteins, e.g., recombinant proteins expressed in host cellsof the invention. The present invention also provides methods forproducing, in vivo, complex glycoprotein products bearing a terminalgalactose, preferably having the structureGal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ as well as hybrid glycoprotein productsbearing a terminal galactose, preferably having the structureGalGlcNAcMan₅GlcNAc₂. The present invention also provides methods forproducing, in vivo, glycoprotein products bearing a terminal sialicacid, preferably having the structure NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ as well as hybrid glycoprotein products bearing aterminal sialic acid, preferably having the structureNANAGalGlcNAcMan₅GlcNAc₂.

The present invention also provides recombinant molecules derived from acombinatorial nucleic acid library; vectors, including expressionvectors, comprising such recombinant molecules; proteins encoded by therecombinant molecules and vectors; host cells transformed with therecombinant molecules or vectors; and glycoproteins produced from suchtransformed hosts.

Further aspects of this invention include methods, compositions and kitsfor diagnostic and therapeutic uses in which the presence or absence ona glycoprotein of Man₅GlcNAc₂, GlcNAcMan₅GlcNAc₂,Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂, GalGlcNAcMan₅GlcNAc₂,NANAGalGlcNAcMan₅GlcNAc₂ and/or NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂may be detected.

In one embodiment, the invention comprises a method for producing arecombinant glycoprotein comprising sialic acid in a non-humaneukaryotic host cell comprising introducing into the host cell a nucleicacid encoding a sialyltransferase enzyme. In certain embodiments, saidhost cell lacks an endogenous sialyltransferase activity. Thesialyltransferase enzyme may be a fusion protein comprising asialyltransferase catalytic domain and a cellular targeting signalpeptide to target the sialyltransferase catalytic domain to thesecretory pathway of the host cell. The cellular targeting signalpeptide may be derived from Mnn2 and may comprise amino acids 1 to 108of GenBank Accession No. (“GenBank AN”) NP_(—)009571.

The invention also comprises a method for producing a recombinantglycoprotein comprising sialic acid in a non-human eukaryotic host cell,comprising introducing into the host cell a nucleic acid encoding atrans-sialidase enzyme. In other embodiments, said nucleic acid encodesa fusion protein comprising a trans-sialidase catalytic domain with acellular targeting signal peptide to target the trans-sialidasecatalytic domain to the secretory pathway, cell wall or plasma membraneof the host cell. In one embodiment, said method further comprisessupplementing the medium for growing the host cell with a sialic aciddonor, such as CMP-Sia. In other embodiments, said method may comprisesupplementing the medium with one or more precursors to a sialic aciddonor. Such precursors may include, for example, one or more ofglucosamine [GlcN], GlcNAc, UDP-GlcNAc, and ManNAc, respectively. In oneembodiment, the method further comprises the step of introducing intothe host cell one or more nucleic acids encoding one or more enzymesinvolved in the biosynthesis or transport of CMP-Sialic acid.

In one embodiment, the invention provides a method for producing arecombinant sialylated glycoprotein in a host cell, the host cellselected or engineered to produce glycoproteins comprising aGal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform, the method comprising the stepof introducing into the host cell a nucleic acid encoding an enzymehaving sialyltransferase activity, the enzyme comprising asialyltransferase catalytic domain and a cellular targeting signalpeptide to target the sialyltransferase catalytic domain to thesecretory pathway of the host cell; wherein, upon passage of therecombinant glycoprotein through the secretory pathway of the host cell,a recombinant glycoprotein comprising aNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform is produced. In oneembodiment, the cellular targeting signal peptide targets thesialyltransferase catalytic domain to a location in the secretorypathway selected from the group consisting of the endoplasmic reticulum,the Golgi apparatus, the trans-Golgi network and secretory vesicles. Inone embodiment, the method further comprises the step of introducinginto the host cell a nucleic acid encoding one or more enzymes involvedin the biosynthesis or transport of CMP-Sia.

In another embodiment, the invention provides a method for producing arecombinant sialylated glycoprotein in a host cell, the host cellselected or engineered to produce glycoproteins comprising aGal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform, the method comprising the stepof introducing into the host cell: (a) a nucleic acid encoding an enzymehaving sialyltransferase activity; and (b) a nucleic acid encoding oneor more enzymes involved in the biosynthesis or transport of CMP-Sia;wherein upon passage of the recombinant glycoprotein through thesecretory pathway of the host cell, a recombinant glycoproteincomprising a NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform isproduced. In one embodiment, wherein the enzyme having sialyltransferaseactivity comprises a sialyltransferase catalytic domain and a cellulartargeting signal peptide to target the sialyltransferase catalyticdomain to the secretory pathway of the host cell.

In another embodiment, the invention comprises a method for producing arecombinant sialylated glycoprotein in a host cell, the host cellselected or engineered to produce glycoproteins comprising aGalGlcNAcMan₅GlcNAc₂ glycoform, the method comprising the step ofintroducing into the host cell a nucleic acid encoding an enzyme havingsialyltransferase activity, the enzyme comprising a sialyltransferasecatalytic domain and a cellular targeting signal peptide to target thesialyltransferase catalytic domain to the secretory pathway of the hostcell; wherein, upon passage of the recombinant glycoprotein through thesecretory pathway of the host cell, a recombinant glycoproteincomprising a NANAGalGlcNAcMan₅GlcNA₂ glycoform is produced.

In another embodiment, the invention comprises a method for producing arecombinant sialylated glycoprotein in a host cell, the host cellselected or engineered to produce glycoproteins comprising aGalGlcNAcMan₅GlcNAc₂ glycoform, the method comprising the step ofintroducing into the host cell: (a) a nucleic acid encoding an enzymehaving sialyltransferase activity; and (b) a nucleic acid encoding oneor more enzymes involved in the biosynthesis or transport of CMP-Sia;wherein upon passage of the recombinant glycoprotein through the Golgiapparatus of the host cell, a recombinant glycoprotein comprising aNANAGalGlcNAcMan₅GlcNA₂ glycoform is produced.

The invention also comprises a method for producing a recombinantglycoprotein comprising sialic acid in a recombinant non-humaneukaryotic host cell comprising introducing into medium used for growingthe host cell a trans-sialidase enzyme and a sialic acid donor.

In other embodiments, the above described methods may further comprisethe step of introducing into the host cell one or more additionalnucleic acids encoding one or more enzymes selected from the groupconsisting of glycosyltransferases, glycosidases and sugar transporters.

In one embodiment, said method produces a recombinant glycoproteincomprising a NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform. Inanother embodiment, said method produces a recombinant glycoproteincomprising a NANAGalGlcNAcMan₅GlcNA₂ glycoform.

In certain embodiments, the host cell to be used in the claimed methodsmay be selected or engineered to comprise a cellular pool of CMP-sialicacid, or may be cultured in the presence of a sialic acid donor, such asCMP-sialic acid, or in the presence of a precursor of a sialic aciddonor. In certain embodiments, the host cell may be selected orengineered to produce CMP-sialic acid. In other embodiments, the hostcell may be modified to express one or more enzyme activities involvedin the CMP-Sia pathway.

In yet other embodiments, the host cell to be used in the claimedmethods may be modified to express one or more glycosylation enzymesselected from the group consisting of glycosyltransferases, glycosidasesand sugar transporters. In a preferred embodiment, the host cell of theclaimed methods has bee modified (selected or engineered) to express aCMP-sialic acid transporter. The glycosylation enzymes may also befusion proteins comprising a catalytic domain and a cellular targetingsignal peptide to target the catalytic domain to a subcellular locationin the host cell.

The host cell to be used in the above described method may be any hostcell. In some embodiments, the host cell lacks endogenoussialyltransferase activity. In some embodiments, the host cell lacksendogenous CMP-Sia. In other embodiments, the host cell is selected fromthe group consisting of a lower eukaryotic host cell, an insect cell ora plant cell. In other embodiments, the host cell is selected from thegroup consisting of any Pichia sp., any Saccharomyces sp., Hansenulapolymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillussp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp. andNeurospora crassa.

In other embodiments, the host cell to be used in the above describedmethods produces a glycoprotein comprising a terminal galactose.

In some embodiments, the host cells to be used in the claimed methodsmay produce glycoproteins comprise a terminal galactose. In someembodiments, the host cell may produce complex glycoproteins comprisinga Gal₍₁₋₄₎GlcNAc₍₁₋₄₎ Man₃GlcNAc₂ glycoform. In other embodiments, thehost cell may produce hybrid glycoproteins comprising aGalGlcNAcMan₅GlcNAc₂ glycoform.

In certain embodiments, the above described method may further comprisethe step of introducing into the host cell which lacks endogenousCMP-Sia one or more additional nucleic acids encoding one or moreenzymes involved in the biosynthesis or transport of CMP-Sialic acid. Inone embodiment, the above described method comprises the step ofintroducing into the host cell one or more additional nucleic acidsencoding one or more mammalian enzymes selected from the groupconsisting of: UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosaminekinase, N-acetylneuraminate-9-phosphate synthase,N-acetylneuraminate-9-phosphatase, CMP-sialic acid phosphatase andCMP-sialic acid synthase. In another embodiment, the above describedmethod comprises the step of introducing into the host cell one or moreadditional nucleic acids encoding one or more bacterial enzymes selectedfrom the group consisting of: UDP-GlcNAc epimerase, sialate synthase andCMP-Sia synthase. In another embodiment, the above described methodcomprises the step of introducing into the host cell one or moreadditional nucleic acids encoding one or more bacterial enzymes selectedfrom the group consisting of: bacterial UDP-GlcNAc epimerase (NeuC),sialate synthase (NeuB) and a mammalian CMP-Sia synthase.

The invention also comprises a nucleic acid encoding a fusion proteincomprising a sialyltransferase catalytic domain and a cellular targetingsignal peptide to target the sialyltransferase catalytic domain to thesecretory pathway of the host cell. For example, suitable cellulartargeting signal peptides may target the sialyltransferase catalyticdomain to the endoplasmic reticulum, the Golgi apparatus, thetrans-Golgi network, or secretory vesicles. In one embodiment, thecellular targeting signal peptide targets the sialyltransferasecatalytic domain to the Golgi apparatus. In one embodiment, saidcellular targeting signal comprises amino acids 1 to 108 of GenBank AN:NP_(—)00971.

The invention also comprises a nucleic acid encoding a fusion proteincomprising a trans-sialidase catalytic domain and a cellular targetingsignal peptide to target the trans-sialidase catalytic domain to thesecretory pathway, the cell wall or cell membrane of the host cell.

The invention also comprises the host cells to be used in the methods ofthe invention, having the characteristics disclosed above in connectionto the claimed methods. In one embodiment, a host cell of the inventioncomprises a non-human eukaryotic host cell that has been geneticallyengineered to express a nucleic acid encoding an enzyme havingsialyltransferase activity or a trans-sialidase activity. In oneembodiment, the host cell of the invention is a non-human eukaryotichost cell that has been genetically engineered to produce CMP-sialicacid. In one embodiment, a host cell of the invention comprises anon-human eukaryotic host cell that has been genetically engineered toexpress a nucleic acid encoding an enzyme having sialyltransferaseactivity or a trans-sialidase activity, and to produce CMP-sialic acid.

In another embodiment, the invention comprises a lower eukaryotic hostcell capable of producing a recombinant glycoprotein comprising acomplex NANA₍₁₋₄ Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform, or a hybridNANAGalGlcNAcMan₅GlcNAc₂, or a NANAGalGlcNAcMan₅GlcNAc₂ glycoform.

The invention also comprises glycoprotein compositions producedaccording to the methods of the invention. In certain embodiments, saidglycoprotein composition may predominantly comprise a complexNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎ Man₃GlcNAc₂ glycoform. In otherembodiments, said glycoprotein composition may comprise a hybridNANAGalGlcNAcMan₅GlcNAc₂.

The invention also comprises glycoprotein compositions producedaccording to the methods of the invention. In certain embodiments, saidglycoprotein compositions predominantly comprise complexNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎ Man₃GlcNAc₂ glycoforms. In otherembodiments, said glycoprotein compositions comprise predominantlyhybrid NANAGalGlcNAcMan₅GlcNAc₂.

The invention also provides a method for producing a sialylatedglycoprotein in a recombinant non-human eukaryotic host cell comprisingintroducing into medium used for growing the host a trans-sialidaseenzyme and a sialic acid donor.

The invention also provides a method of enhancing the production ofCMP-sialic acid in a host cell comprising the step of introducing intothe host cell a nucleic acid encoding an enzyme having GlcNAc epimeraseactivity.

The invention also provides a method of enhancing the production ofCMP-sialic acid in a host cell comprising the step of introducing intothe host cell a nucleic acid encoding an enzyme having sialate aldolaseactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a typical fungal N-glycosylationpathway.

FIG. 1B is a schematic diagram of a typical human N-glycosylationpathway.

FIG. 2 depicts construction of a combinatorial DNA library of fusionconstructs. Panel A diagrams the insertion of a targeting peptidefragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). Panel B showsthe generated targeting peptide sub-library having restriction sitesNotI-AscI. Panel C diagrams the insertion of a catalytic domain regioninto pJN347, a modified pUC19 vector. Panel D shows the generatedcatalytic domain sub-library having restriction sites NotI, AscI andPacI. Panel E depicts one particular fusion construct generated from thetargeting peptide sub-library and the catalytic domain sub-library.

FIG. 3 (SEQ ID NOS: 45-46 respectively, in order of appearance)illustrates the M. musculus α-1,2-mannosidase IA open reading frame. Thesequences of the PCR primers used to generate N-terminal truncations areunderlined.

FIGS. 4A-4F illustrates engineering of vectors with multiple auxotrophicmarkers and genetic integration of target proteins in the P. pastorisOCH1 locus.

FIGS. 5A-5E show MALDI-TOF analysis demonstrating production of kringle3 domain of human plasminogen (K3) glycoproteins having Man₅GlcNAc₂ asthe predominant N-glycan structure in P. pastoris. FIG. 5A depicts thestandard Man₅GlcNAc₂ [a] glycan (Glyko, Novato, Calif.) andMan₅GlcNAc₂+Na⁺ [b]. FIG. 5B shows PNGase—released glycans from K3 wildtype. The N-glycans shown are as follows: Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]. FIG. 5C depicts the och1deletion resulting in the production of Man₈GlcNAc₂ [c] as thepredominant N-glycan. FIGS. 5D and 5E show the production of Man₅GlcNAc₂[b] after in vivo trimming of Man₈GlcNAc₂ with a chimericα-1,2-mannosidase. The predominant N-glycan is indicated by a peak witha mass (m/z) of 1253 consistent with its identification as Man₅GlcNAc₂[b].

FIGS. 6A-6F show MALDI-TOF analysis demonstrating production of IFN-βglycoproteins having Man₅GlcNAc₂ as the predominant N-glycan structurein P. pastoris. FIG. 6A shows the standard Man₅GlcNAc₂ [a] andMan₅GlcNAc₂+Na⁺ [b] as the standard (Glyko, Novato, Calif.). FIG. 6Bshows PNGase—released glycans from IFN-β wildtype. FIG. 6C depicts theoch1 knock-out producing Man₈GlcNAc₂ [c]; Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂[g]; and no production of Man₅GlcNAc₂[b]. FIG. 6D shows relatively small amount of Man₅GlcNAc₂ [b] amongother intermediate N-glycans Man₈GlcNAc₂[c] to Man₁₂GlcNAc₂ [g]. FIG. 6Eshows a significant amount of Man₅GlcNAc₂ [b] relative to the otherglycans Man₈GlcNAc₂[c] and Man₉GlcNAc₂[d] produced by pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99). FIG. 6F showspredominant production of Man₅GlcNAc₂[b] on the secreted glycoproteinIFN-β by pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187). TheN-glycan is indicated by a peak with a mass (m/z) of 1254 consistentwith its identification as Man₅GlcNAc₂ [b].

FIG. 7 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pFB8 mannosidase, which demonstratesa lack of extracellular mannosidase activity in the supernatant; and (C)Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reeseimannosidase (positive control).

FIG. 8 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pGC5 mannosidase, which demonstratesa lack of extracellular mannosidase activity in the supernatant; and (C)Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reeseimannosidase (positive control).

FIG. 9 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pBC18-5 mannosidase, whichdemonstrates lack of extracellular mannosidase activity in thesupernatant; and (C) supernatant of medium P. pastoris, Δoch1transformed with pDD28-3, which demonstrates activity in the supernatant(positive control).

FIGS. 10A-10B demonstrate the activity of an UDP-GlcNAc transporter inthe production of GlcNAcMan₅GlcNAc₂ in P. pastoris. FIG. 10A depicts aP. pastoris strain (YSH-3) with a human GnTI but without the UDP-GlcNActransporter resulting in some production of GlcNAcMan₅GlcNAc₂ [b] but apredominant production of Man₅GlcNAc₂ [a]. FIG. 10B depicts the additionof UDP-GlcNAc transporter from K. lactis in a strain (PBP-3) with thehuman GnTI, which resulted in the predominant production ofGlcNAcMan₅GlcNAc₂ [b]. The single prominent peak of mass (m/z) at 1457is consistent with its identification as GlcNAcMan₅GlcNAc₂ [b] as shownin FIG. 10B.

FIG. 11 shows a pH optimum of a heterologous mannosidase enzyme encodedby pBB27-2 (Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31)expressed in P. pastoris.

FIGS. 12A-12C show MALDI-TOF analysis of N-glycans released from a cellfree extract of K. lactis. FIG. 12A shows the N-glycans released fromwild-type cells, which includes high-mannose type N-glycans. FIG. 12Bshows the N-glycans released from och1 mnn1 deleted cells, revealing adistinct peak of mass (m/z) at 1908 consistent with its identificationas Man₉GlcNAc₂ [d]. FIG. 12C shows the N-glycans released from och1 mnn1deleted cells after in vitro α-1,2-mannosidase digest corresponding to apeak consistent with Man₅GlcNAc₂.

FIG. 13 represents T-DNA cassettes with catalytic domain(s) ofglycosylation enzymes fused in-frame to different leader sequences. Theends of the T-DNA are marked by the right (rb) and left borders (lb).Various promoters and terminators may also be used. The plant selectablemarker can also be varied. The right and left borders are required onlyfor agrobacterium-mediated transformation and not for particlebombardment or electroporation.

FIG. 14 illustrates the CMP-sialic acid biosynthetic pathway in mammalsand bacteria. Enzymes involved in each pathway are italicized. Theprimary substrates, intermediates and products are in bold. (PEP:phosphoenol pyruvate; CTP: cytidine triphosphate).

FIG. 15 shows the open reading frame (ORF) of E. coli protein NeuC(Genbank AN: M84026.1; SEQ ID NO: 57) and the predicted amino acidsequence (SEQ ID NO:58). The underlined DNA sequences are regions towhich primers have been designed to amplify the ORF.

FIG. 16 shows the ORF of E. coli protein NeuB (Genbank AN: U05248.1; SEQID NO:59) and the predicted amino acid sequence (SEQ ID NO:60). Theunderlined DNA sequences are regions to which primers have been designedto amplify the ORF.

FIG. 17 shows the ORF of E. coli protein NeuA (Genbank AN: J05023.1; SEQID NO:61) and the predicted amino acid sequence (SEQ ID NO:62). Theunderlined DNA sequences are regions to which primers have been designedto amplify the ORF.

FIG. 18 shows the ORF of Mus musculus CMP-Sia synthase (Genbank AN:AJ006215; SEQ ID NO:63) and the amino acid sequence (SEQ ID NO:64). Theunderlined DNA sequences are regions to which primers have been designedto amplify the ORF.

FIG. 19 illustrates an alternative biosynthetic route for generatingN-acetylmannosamine (ManNAc) in vivo. Enzymes involved in each pathwayare italicized. The primary substrates, intermediates and products arein bold.

FIG. 20 shows the ORF of Sus scrofa GlcNAc epimerase (Genbank AN:D83766; SEQ ID NO:65) and the amino acid sequence (SEQ ID NO:66). Theunderlined DNA sequences are regions to which primers have been designedto amplify the ORF.

FIG. 21 illustrates the reversible reaction catalyzed by sialatealdolase and its dependence on sialic acid (Sia) concentration. Enzymesinvolved in each pathway are italicized. The primary substrates,intermediates and products are in bold.

FIG. 22 shows the ORF of E. coli sialate aldolase (Genbank AN: X03345;SEQ ID NO:67) and the amino acid sequence (SEQ ID NO:68). The underlinedDNA sequences are regions to which primers have been designed to amplifythe ORF.

FIG. 23 shows a HPLC of negative control of cell extracts from strainYSH99a incubated under assay conditions (Example 16) in the absence ofacceptor glycan. The doublet peak eluting at 26.5 min results fromcontaminating cellular component(s).

FIG. 24 shows a HPLC of positive control cell extract from strain YSH99aincubated under assay conditions (Example 16) in the presence of 2-AB(aminobenzamide) labeled acceptor glycan and supplemented withCMP-sialic acid. The peak eluting at 23 min corresponds to sialylationon each branch of a biantennary galactosylated N-glycan. The doubletpeak eluting at 26.5 min results from contaminating cellularcomponent(s).

FIG. 25 shows a HPLC of a cell extract from strain YSH99a incubatedunder assay conditions (Example 16) in the presence of acceptor glycanwith no exogenous CMP-sialic acid. The peaks eluting at 20 and 23 mincorrespond to mono- and di-sialylation of a biantennary galactosylatedN-glycan. The doublet peak eluting at 26.5 min results fromcontaminating cellular component(s).

FIG. 26 shows sialidase treatment of N-glycans from YSH99a extractincubation. The sample illustrated in FIG. 25 was incubated overnight at37° C. in the presence of 100 U sialidase (New England Biolabs,Beverley, Mass.). The peaks eluting at 20 and 23 min corresponding tomono- and di-sialylated N-glycan, have been removed in FIG. 25. Thecontaminating peak at 26 min remains.

FIG. 27 shows commercial mono- and di-sialylated N-glycan standards. Thepeaks eluting at 20 and 23 min correspond to mono- and di-sialylation ofthe commercial standards A1 and A2 (Glyko Inc., San Rafael, Calif.).

FIG. 28 illustrates vector pSH321b used in Example 17.

FIG. 29 depicts vector pJN711b used in Example 17.

FIG. 30 depicts vector pSH326b used in Example 17.

FIG. 31 depicts vector pSH370 used in Example 17.

FIG. 32 depicts vector pSH373 used in Example 17.

FIG. 33 shows the nucleic acid (SEQ ID NO:101) and amino acid sequence(SEQ ID NO:102) of the codon-optimized hST6Gal leader 53 fusiondescribed in Example 17.

FIG. 34 depicts vector pSH568 used in Example 17.

FIG. 35 depicts MALDI-TOF MS analysis of the glycans produced in YSH126and YSH145. The “+ve” sign at the upper right corner of each MALDI-ROFindicates analysis of positive ions and the “−ve” sign indicatesanalysis of negative ions.

FIG. 36 shows MALDI-TOF MS analysis of the glycans produced in YSH145and YSH160.

FIG. 37 shows MALDI-TOF MS analysis of the glycans produced in YSH272.The solid arrows represent sialic acid containing glycans. The dashedarrows represent neutral glycans.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology,Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.;Handbook of Biochemistry: Section A Proteins Vol I 1976 CRC Press;Handbook of Biochemistry: Section A Proteins Vol II 1976 CRC Press;Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).The nomenclatures used in connection with, and the laboratory proceduresand techniques of, molecular and cellular biology, protein biochemistry,enzymology and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

All publications, patents and other references mentioned herein areincorporated by reference.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “N-glycan” refers to an N-linkedoligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂(“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannosecore” used with respect to the N-glycan also refers to the structureMan₃GlcNAc₂ (“Man₃”). N-glycans differ with respect to the number ofbranches (antennae) comprising peripheral sugars (e.g., fucose andsialic acid) that are added to the Man₃ core structure. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid).

A “high mannose” type N-glycan has five or more mannose residues. A“complex” type N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of the trimannose core. Complex N-glycans may also have galactose(“Gal”) residues that are optionally modified with sialic acid orderivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac”refers to acetyl). A complex N-glycan typically has at least one branchthat terminates in an oligosaccharide such as, for example: NeuNAc-;NeuAca2-6GalNAca1-; NeuAca2-3Galb1-3GalNAca1-;NeuAca2-3/6Galb1-4GlcNAcb1-; GlcNAca1-4Galb1-(mucins only);Fuca1-2Galb1-(blood group H). Sulfate esters can occur on galactose,GalNAc, and GlcNAc residues, and phosphate esters can occur on mannoseresidues. NeuAc (Neu: neuraminic acid; Ac:acetyl) can be O-acetylated orreplaced by NeuGl (N-glycolylneuraminic acid). Complex N-glycans mayalso have intrachain substitutions comprising “bisecting” GlcNAc andcore fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on theterminal of the 1,3 mannose arm of the trimannose core and zero or moremannoses on the 1,6 mannose arm of the trimannose core.

The term “homologs” used with respect to an original enzyme or gene of afirst family or species refers to distinct enzymes or genes of a secondfamily or species which are determined by functional, structural orgenomic analyses to be an enzyme or gene of the second family or specieswhich corresponds to the original enzyme or gene of the first family orspecies. Most often, homologs will have functional, structural orgenomic similarities. Techniques are known by which homologs of anenzyme or gene can readily be cloned using genetic probes and PCR.Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

The term “predominant” or “predominantly” used with respect to theproduction of N-glycans refers to a structure which represents the majorpeak detected by matrix assisted laser desorption ionization time offlight mass spectrometry (MALDI-TOF) analysis.

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAcTr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes;“NANA” refers to N-acetylneuraminic acid.

As used herein, a “humanized glycoprotein” or a “human-likeglycoprotein” refers alternatively to a protein having attached theretoN-glycans having less than four mannose residues, and syntheticglycoprotein intermediates (which are also useful and can be manipulatedfurther in vitro or in vivo) having at least five mannose residues.Preferably, glycoproteins produced according to the invention contain atleast 30 mole %, preferably at least 40 mole % and more preferably50-100 mole % of the Man₅GlcNAc₂ intermediate, at least transiently.This may be achieved, e.g., by engineering a host cell of the inventionto express a “better”, i.e., a more efficient glycosylation enzyme. Forexample, a mannosidase is selected such that it will have optimalactivity under the conditions present at the site in the host cell whereproteins are glycosylated and is introduced into the host cellpreferably by targeting the enzyme to a host cell organelle whereactivity is desired.

The term “enzyme”, when used herein in connection with altering hostcell glycosylation, refers to a molecule having at least one enzymaticactivity, and includes full-length enzymes, catalytically activefragments, chimerics, complexes, and the like. A “catalytically activefragment” of an enzyme refers to a polypeptide having a detectable levelof functional (enzymatic) activity.

A “lower eukaryotic host cell,” when used herein in connection withglycosylation profiles, refers to any eukaryotic cell which ordinarilyproduces high mannose containing N-glycans, and thus is meant toinclude, in a fuctional sense, some animal or plant cells whoseglycosylation profiles are like those of lower eukaryotic cells, andmost typical lower eukaryotic cells, including uni- and multicellularfungal and algal cells.

As used herein, the term “secretion pathway” refers to the assembly lineof various glycosylation enzymes to which a lipid-linked oligosaccharideprecursor and an N-glycan substrate are sequentially exposed, followingthe molecular flow of a nascent polypeptide chain from the cytoplasm tothe endoplasmic reticulum (ER) and the compartments of the Golgiapparatus. The term “secretory pathway” thus refers to organelles andcomponents within the cell where glycoproteins are modified inpreparation for secretion. The secretory pathway includes theendoplasmic reticulum or ER, the Golgi apparatus, the trans-Golginetwork and the secretory vesicles. Enzymes are said to be localizedalong this pathway. An enzyme X that acts on a lipid-linked glycan or anN-glycan before enzyme Y is said to be or to act “upstream” to enzyme Y;similarly, enzyme Y is or acts “downstream” from enzyme X.

The term “targeting peptide” α “targeting signal peptide” as used hereinrefers to nucleotide or amino acid sequences encoding a cellulartargeting signal peptide which mediates the localization (or retention)of an associated sequence to sub-cellular locations, e.g., organelles.

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation. The term includes single and double strandedforms of DNA. A nucleic acid molecule of this invention may include bothsense and antisense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. They may be modified chemicallyor biochemically or may contain non-natural or derivatized nucleotidebases, as will be readily appreciated by those of skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.) Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X”refers to a nucleic acid, at least a portion of which has either (i) thesequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ IDNO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

An “isolated” or “substantially pure” nucleic acid or polynucleotide(e.g., an RNA, DNA or a mixed polymer) is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases, and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acidor polynucleotide so described has itself been physically removed fromits native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence (i.e., a sequence that is not naturally adjacentto this endogenous nucleic acid sequence) is placed adjacent to theendogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. By way of example, anon-native promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of ahuman cell, such that this gene has an altered expression pattern. Thisgene would now become “isolated” because it is separated from at leastsome of the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site, a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, 1990, herebyincorporated herein by reference). For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, hereby incorporated herein by reference.

The terms “significant alignment”, “substantial homology” or“substantial similarity,” when referring to a nucleic acid or fragmentthereof, indicates that, when optimally aligned with appropriatenucleotide insertions or deletions with another nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 50%, more preferably 60% of the nucleotide bases, usually at leastabout 70%, more usually at least about 80%, preferably at least about90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of thenucleotide bases, as measured by any well-known algorithm of sequenceidentity, such as FASTA, BLAST or Gap, as discussed above.

Alternatively, significant alignment, substantial homology or similarityexists when a nucleic acid or fragment thereof hybridizes to anothernucleic acid, to a strand of another nucleic acid, or to thecomplementary strand thereof, under stringent hybridization conditions.“Stringent hybridization conditions” and “stringent wash conditions” inthe context of nucleic acid hybridization experiments depend upon anumber of different physical parameters. Nucleic acid hybridization willbe affected by such conditions as salt concentration, temperature,solvents, the base composition of the hybridizing species, length of thecomplementary regions, and the number of nucleotide base mismatchesbetween the hybridizing nucleic acids, as will be readily appreciated bythose skilled in the art. One having ordinary skill in the art knows howto vary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., supra, page 9.51, herebyincorporated herein by reference. For purposes herein, “high stringencyconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled artisan that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

The nucleic acids (also referred to as polynucleotides) of thisinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung, 1989; Caldwell, 1992; and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest. See, e.g., Reidhaar-Olson 1988).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”).

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that has been geneticallyengineered. A recombinant host cell includes a cell into which arecombinant vector has been introduced. It should be understood thatsuch terms are intended to refer not only to the particular subject cellbut to the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism. The term “host” refers to any organism, animal or plant,comprising one or more “host cells”, or to the source of the “hostcells”.

Moreover, as used herein a “host cell which lacks endogenous CMP-Sia”refers to a cell that does not endogeneously produce CMP-Sia, includingcells which lack a CMP-Sia pathway. As used herein, a “fungal host cell”refers to a fungal host cell that lacks CMP-Sia.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” as used herein encompasses bothnaturally-occurring and non-naturally-occurring proteins, and fragments,mutants, derivatives and analogs thereof. A polypeptide may be monomericor polymeric. Further, a polypeptide may comprise a number of differentdomains each of which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) when it exists in a purity not found in nature,where purity can be adjudged with respect to the presence of othercellular material (e.g., is free of other proteins from the samespecies) (3) is expressed by a cell from a different species, or (4)does not occur in nature (e.g., it is a fragment of a polypeptide foundin nature or it includes amino acid analogs or derivatives not found innature or linkages other than standard peptide bonds). Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A polypeptide orprotein may also be rendered substantially free of naturally associatedcomponents by isolation, using protein purification techniqueswell-known in the art. As thus defined, “isolated” does not necessarilyrequire that the protein, polypeptide, peptide or oligopeptide sodescribed has been physically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion compared toa full-length polypeptide. In a preferred embodiment, the polypeptidefragment is a contiguous sequence in which the amino acid sequence ofthe fragment is identical to the corresponding positions in thenaturally-occurring sequence. Fragments typically are at least 5, 6, 7,8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 aminoacids long, more preferably at least 20 amino acids long, morepreferably at least 25, 30, 35, 40 or 45, amino acids, even morepreferably at least 50 or 60 amino acids long, and even more preferablyat least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labeling polypeptides andof substituents or labels useful for such purposes are well-known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well-known in the art. See Ausubelet al., 1992, hereby incorporated by reference.

A “polypeptide mutant” or “mutein” or “variant” refers to a polypeptidewhose sequence contains an insertion, duplication, deletion,rearrangement or substitution of one or more amino acids compared to theamino acid sequence of a native or wild type protein. A mutein may haveone or more amino acid point substitutions, in which a single amino acidat a position has been changed to another amino acid, one or moreinsertions and/or deletions, in which one or more amino acids areinserted or deleted, respectively, in the sequence of thenaturally-occurring protein, and/or truncations of the amino acidsequence at either or both the amino or carboxy termini. A mutein mayhave the same but preferably has a different biological activitycompared to the naturally-occurring protein.

A mutein has at least 70% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having 80%, 85% or 90%overall sequence homology to the wild-type protein. In an even morepreferred embodiment, a mutein exhibits 95% sequence identity, even morepreferably 97%, even more preferably 98% and even more preferably 99%overall sequence identity. Sequence homology may be measured by anycommon sequence analysis algorithm, such as Gap or Bestfit.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, and other unconventional amino acids may also besuitable components for polypeptides of the present invention. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, s-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences). In a preferred embodiment, a homologousprotein is one that exhibits 60% sequence homology to the wild typeprotein, more preferred is 70% sequence homology. Even more preferredare homologous proteins that exhibit 80%, 85% or 90% sequence homologyto the wild type protein. In a yet more preferred embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence identity. Asused herein, homology between two regions of amino acid sequence(especially with respect to predicted structural similarities) isinterpreted as implying similarity in function.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, hereby incorporated herein by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a inhibitory molecule sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996;Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul,1997). Preferred parameters for BLASTp are: Expectation value: 10(default); Filter: seg (default); Cost to open a gap: 11 (default); Costto extend a gap: 1 (default); Max. alignments: 100 (default); Word size:11 (default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, hereby incorporated herein by reference). For example, percentsequence identity between amino acid sequences can be determined usingFASTA with its default parameters (a word size of 2 and the PAM250scoring matrix), as provided in GCG Version 6.1, hereby incorporatedherein by reference.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusion proteins can beproduced recombinantly by constructing a nucleic acid sequence whichencodes the polypeptide or a fragment thereof in-frame with a nucleicacid sequence encoding a different protein or peptide and thenexpressing the fusion protein. Alternatively, a fusion protein can beproduced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

“Specific binding” refers to the ability of two molecules to bind toeach other in preference to binding to other molecules in theenvironment. Typically, “specific binding” discriminates overadventitious binding in a reaction by at least two-fold, more typicallyby at least 10-fold, often at least 100-fold. Typically, the affinity oravidity of a specific binding reaction is at least about 10⁻⁷ M (e.g.,at least about 10⁻⁸ M or 10⁻⁹ M).

As used herein, a “CMP-Sialic acid biosynthetic pathway” or a “CMP-Siabiosynthetic pathway” refers to one or more glycosylation enzymes whichresults in the formation of CMP-Sia in a host.

As used herein, a “CMP-Sia pool” refers to a detectable level ofcellular CMP-Sia. The CMP-Sia pool may be the result of the productionof CMP-Sia by the host cell, or of the uptake of CMP-Sia from theculture media.

The substrate UDP-GlcNAc is the abbreviation forUDP-N-acetylglucosamine. The intermediate ManNAc is the abbreviation forN-acetylmannosamine. The intermediate ManNAc-6-P is the abbreviation forN-acetylmannosamine-6-phosphate. The intermediate Sia-9-P is theabbreviation for sialate-9-phosphate. The intermediate Cytidinemonophosphate-sialic acid is abbreviated as “CMP-Sia.” Sialic acid isabbreviated as “Sia,” “Neu5Ac,” “NeuAc” or “NANA” herein.

As used herein, the term “sialic acid” refers to a group of moleculeswhere the common molecule includes N-acetyl-5-neuraminic acid (Neu5Ac)having the basic 9-carbon neuraminic acid core modified at the 5-carbonposition with an attached acetyl group. Common derivatives of Neu5Ac atthe 5-carbon position include: 2-keto-3-deoxy-d-glycero-d-galactonononicacid (KDN) which possesses a hydroxyl group in place of the acetylgroup; de-N-acetylation of the 5-N-acetyl group produces neuraminic(Neu); hydroxylation of the 5-N-acetyl group producesN-glycolylneuraminic acid (Neu5Gc). The hydroxyl groups at positions 4-,7-, 8- and 9- of these four molecules (Neu5Ac, KDN, Neu and Neu5Gc) canbe further substituted with 0-acetyl, O-methyl, 0-sulfate and phosphategroups to enlarge this group of compounds. Furthermore, unsaturated anddehydro forms of sialic acids are known to exist.

The gene encoding for the UDP-GlcNAc epimerase is abbreviated as “NeuC.”The gene encoding for the sialate synthase is abbreviated as “NeuB.” Thegene encoding for the CMP-Sialate synthase is abbreviated as “NeuA.”

Sialate aldolase is also commonly referred to as sialate lyase andsialate pyruvate-lyase. More specifically in E. coli, sialate aldolaseis referred to as NanA. See Ringerberg et al. (2001).

As used herein, the term “sialic acid donor” refers to a molecule orentity that is capable of providing a sialic acid residue for transferto a substrate by enzymatic or synthetic means. A sialic acid donor canbe a sugar nucleotide precursor such as CMP-Sia. In certain embodimentsof the invention, a precursor to a sialic acid donor may be used, inconjunction with adapted host cells which are competent for conversionof the precursor moleculeto the sialic acid donor. Such adapted hostcells may, for example have be capable of producing one or more sugarnucleotide precursors of CMP-Sia such as UDP-GlcNAc [glucosamine] andManNAc. Thus the “sialic acid donor” may be provided by providing anadapted host cell which contains one or more sugar nucleotideprecursors, such as UDP-GlcNAc or ManNAc, and which has functionalenzyme activity for the conversion of these molecules to CMP-Sialicacid. The host cell may have such enzyme activity endogenously, or maybe engineered to express enzymes which are able to convert UDP-GlcNAc orManNAc to CMP-Sia. In other embodiments, the host cell may be culturedin media containing the necessary enzymes for the conversion of theprecursor molecule to the sialic acid donor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting. Throughout this specification and claims, the word “comprise”or variations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Methods for Producing Host Cells Having Man₅GlcNAc₂ ModifiedOligosaccharides for the Generation of Human-Like N-Glycans

The invention provides a method for producing a glycoprotein havinghuman-like glycosylation in a non-human eukaryotic host cell. Asdescribed in more detail below, a eukaryotic host cell that does notnaturally express, or which is engineered not to express, one or moreenzymes involved in production of high mannose structures is selected asa starting host cell. Such a selected host cell is engineered to expressone or more enzymes or other factors required to produce human-likeglycoproteins. A desired host strain can be engineered one enzyme ormore than one enzyme at a time. In addition, a nucleic acid moleculeencoding one or more enzymes or activities may be used to engineer ahost strain of the invention. Preferably, a library of nucleic acidmolecules encoding potentially useful enzymes (e.g., chimeric enzymescomprising a catalytically active enzyme fragment ligated in-frame to aheterologous subcellular targeting sequence) is created (e.g., byligation of sub-libraries comprising enzymatic fragments and subcellulartargeting sequences), and a strain having one or more enzymes withoptimal activities or producing the most “human-like” or otherwisedesirable glycoproteins may be selected by transforming target hostcells with one or more members of the library.

In particular, the methods described herein enable one to obtain, invivo, Man₅GlcNAc₂ structures in high yield, at least transiently, forthe purpose of further modifying it to yield complex N-glycans. Asuccessful scheme to obtain suitable Man₅GlcNAc₂ structures inappropriate yields in a host cell, such as a lower eukaryotic organism,generally involves two parallel approaches: (1) reducing high mannosestructures made by endogenous mannosyltransferase activities, if any,and (2) removing 1,2-α-mannose by mannosidases to yield high levels ofsuitable Man₅GlcNAc₂ structures which may be further reacted inside thehost cell to form complex, human-like glycoforms.

Accordingly, a first step involves the selection or creation of aeukaryotic host cell, e.g., a lower eukaryote, capable of producing aspecific precursor structure of Man₅GlcNAc₂ that is able to accept invivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In oneembodiment, the method involves making or using a non-human eukaryotichost cell depleted in a 1,6 mannosyltransferase activity with respect tothe N-glycan on a glycoprotein. Preferably, the host cell is depleted inan initiating 1,6 mannosyltransferase activity such as an alpha 1,6mannosyltransferase activity (see below). Such a host cell will lack oneor more enzymes involved in the production of high mannose structureswhich are undesirable for producing human-like glycoproteins.

One or more enzyme activities are then introduced into such a host cellto produce N-glycans within the host cell characterized by having atleast 30 mol % of Man₅GlcNAc₂ (“Man₅”) carbohydrate structures.Man₅GlcNAc₂ structures are necessary for complex N-glycan formation:Man₅GlcNAc₂ must be formed in vivo in a high yield (e.g., in excess of30%), at least transiently, as subsequent mammalian- and human-likeglycosylation reactions require Man₅GlcNAc₂ or a derivative thereof.

This step also requires the formation of a particular isomeric structureof Man₅GlcNAc₂ within the cell at a high yield. While Man₅GlcNAc₂structures are necessary for complex N-glycan formation, their presenceis by no means sufficient. That is because Man₅GlcNAc₂ may occur indifferent isomeric forms, which may or may not serve as a substrate forGlcNAc transferase I. As most glycosylation reactions are not complete,a particular glycosylated protein generally contains a range ofdifferent carbohydrate structures (i.e. glycoforms) on its surface.Thus, the mere presence of trace amounts (i.e., less than 5%) of aparticular structure like Man₅GlcNAc₂ is of little practical relevancefor producing mammalian- or human-like glycoproteins. It is theformation of a GlcNAc transferase I-accepting Man₅GlcNAc₂ intermediate(FIG. 1B) in high yield (i.e., above 30%), which is required. Theformation of this intermediate is necessary to enable subsequent in vivosynthesis of complex N-glycans on glycosylated proteins of interest(target proteins).

Accordingly, some or all of the Man₅GlcNAc₂ produced by the selectedhost cell must be a productive substrate for enzyme activities along amammalian glycosylation pathway, e.g., can serve as a substrate for aGlcNAc transferase I activity in vivo, thereby forming the human-likeN-glycan intermediate GlcNAcMan₅GlcNAc₂ in the host cell. In a preferredembodiment, at least 10%, more preferably at least 30% and mostpreferably 50% or more of the Man₅GlcNAc₂ intermediate produced in thehost cell of the invention is a productive substrate for GnTI in vivo.It is understood that if, for example, GlcNAcMan₅GlcNAc₂ is produced at10% and Man₅GlcNAc₂ is produced at 25% on a target protein, the totalamount of transiently produced Man₅GlcNAc₂ is 35% becauseGlcNAcMan₅GlcNAc₂ is a product of Man₅GlcNAc₂.

As described later herein, N-glycans having a terminal GlcNAc residue,such as GlcNAcMan₅GlcNAc₂ can serve as a substrate for sequentialaddition of galactose [e.g., UDP-galactosyltransferase] and sialic acid[e.g., sialyltransferase, CMP-Sia] to produce the hybrid N-glycans ofthe present invention.

One of ordinary skill in the art can select host cells from nature,e.g., existing fungi or other lower eukaryotes that produce significantlevels of Man₅GlcNAc₂ in vivo. As yet, however, no lower eukaryote hasbeen shown to provide such structures in vivo in excess of 1.8% of thetotal N-glycans (see e.g. Maras, 1997). Alternatively, such host cellsmay be genetically engineered to produce the Man₅GlcNAc₂ structure invivo. Methods such as those described in U.S. Pat. No. 5,595,900 may beused to identify the absence or presence of particularglycosyltransferases, mannosidases and sugar nucleotide transporters ina target host cell or organism of interest.

Inactivation of Undesirable Host Cell Glycosylation Enzymes

The methods of the invention are directed to making host cells thatproduce glycoproteins having altered, and preferably human-like,N-glycan structures. In a preferred embodiment, the methods are directedto making host cells in which oligosaccharide precursors are enriched inMan₅GlcNAc₂. Preferably, a eukaryotic host cell is used that does notexpress one or more enzymes involved in the production of high mannosestructures. Such a host cell may be found in nature or may beengineered, e.g., starting with or derived from one of many such mutantsalready described in yeasts. Thus, depending on the selected host cell,one or a number of genes that encode enzymes known to be characteristicof non-human glycosylation reactions may have to be deleted. Such genesand their corresponding proteins have been extensively characterized ina number of lower eukaryotes (e.g., S. cerevisiae, T. reesei, A.nidulans etc.), thereby providing a list of known glycosyltransferasesin lower eukaryotes, their activities and their respective geneticsequence. These genes are likely to be selected from the group ofmannosyltransferases e.g. 1,3 mannosyltransferases (e.g. MNN1 in S.cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KREfamily from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S.cerevisiae), mannosylphosphate transferases and their regulators (MNN4and MNN6 from S. cerevisiae) and additional enzymes that are involved inaberrant, i.e. non human, glycosylation reactions. Many of these geneshave in fact been deleted individually giving rise to viable phenotypeswith altered glycosylation profiles. Examples are shown in Table 1.

Preferred lower eukaryotic host cells of the invention, as describedherein to exemplify the required manipulation steps, arehypermannosylation-minus (och1) mutants of Pichia pastoris or K. lactis.Like other lower eukaryotes, P. pastoris processes Man₉GlcNAc₂structures in the ER with an α-1,2-mannosidase to yield Man₈GlcNAc₂(FIG. 1A). Through the action of several mannosyltransferases, thisstructure is then converted to hypermannosylated structures(Man_(>9)GlcNAc₂), also known as mannans. In addition, it has been foundthat P. pastoris is able to add non-terminal phosphate groups, throughthe action of mannosylphosphate transferases, to the carbohydratestructure. This differs from the reactions performed in mammalian cells,which involve the removal rather than addition of mannose sugars. It isof particular importance to eliminate the ability of the eukaryotic hostcell, e.g., fungus, to hypermannosylate an existing Man₈GlcNAc₂structure. This can be achieved by either selecting for a host cell thatdoes not hypermannosylate or by genetically engineering such a cell.

Genes that are involved in the hypermannosylation process have beenidentified, e.g., in Pichia pastoris, and by creating mutations in thesegenes, one can reduce the production of “undesirable” glycoforms. Suchgenes can be identified by homology to existing mannosyltransferases ortheir regulators (e.g., OCH1, MNN4, MNN6, MNN1) found in other lowereukaryotes such as C. albicans, Pichia angusta or S. cerevisiae or bymutagenizing the host strain and selecting for a glycosylation phenotypewith reduced mannosylation. Based on homologies amongst knownmannosyltransferases and mannosylphosphate transferases, one may eitherdesign PCR primers (SEQ ID NOS: 7, 8, 47 and 4 left to right,respectively, in order of appearance) (examples of which are shown inTable 2), or use genes or gene fragments encoding such enzymes as probesto identify homologs in DNA libraries of the target or a relatedorganism. Alternatively, one may identify a functional homolog havingmannosyltransferase activity by its ability to complement particularglycosylation phenotypes in related organisms.

TABLE 2 PCR Primers Target Gene(s) in PCR primer A PCR primer BP.pastoris Homologs ATGGCGAAGGCAG TTAGTCCTTCCAAC 1,6- OCH1 S.cerevisiae,ATGGCAGT  TTCCTTC  mannosyltransferase Pichia albicans (SEQ ID NO: 1)(SEQ ID NO: 2) TAYTGGMGNGTNG GCRTCNCCCCANCK 1,2 KTR/KRE family,ARCYNGAYATHAA YTCRTA  mannosyltransferases S.cerevisiae (SEQ ID NO: 3)(SEQ ID NO: 4) Legend: M = A or C, R = A or G, W = A or T, S = C or G, Y= C or T, K = G or T, V = A or C or G, H = A or C or T, D = A or G or T,B = C or G or T, N = G or A or T or C.

To obtain the gene or genes encoding 1,6-mannosyltransferase activity inP. pastoris, for example, one would carry out the following steps: OCH1mutants of S. cerevisiae are temperature sensitive and are slow growersat elevated temperatures. One can thus identify functional homologs ofOCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiaewith a P. pastoris DNA or cDNA library. Mutants of S. cerevisiae areavailable, e.g., from Stanford University and are commercially availablefrom ResGen, an Invitrogen Corp. (Carlsbad, Calif.). Mutants thatdisplay a normal growth phenotype at elevated temperature, after havingbeen transformed with a P. pastoris DNA library, are likely to carry anOCH1 homolog of P. pastoris. Such a library can be created by partiallydigesting chromosomal DNA of P. pastoris with a suitable restrictionenzyme and, after inactivating the restriction enzyme, ligating thedigested DNA into a suitable vector, which has been digested with acompatible restriction enzyme.

Suitable vectors include, e.g., pRS314, a low copy (CEN6/ARS4) plasmidbased on pBluescript containing the Trp1 marker (Sikorski, 1989) andpFL44S, a high copy (2μ) plasmid based on a modified pUC19 containingthe URA3 marker (Bonneaud, 1991). Such vectors are commonly used byacademic researchers and similar vectors are available from a number ofdifferent vendors (e.g., Invitrogen (Carlsbad, Calif.); Pharmacia(Piscataway, N.J.); New England Biolabs (Beverly, Mass.)). Furtherexamples include pYES/GS, 2μ origin of replication based yeastexpression plasmid from Invitrogen, or Yep24 cloning vehicle from NewEngland Biolabs.

After ligation of the chromosomal DNA and the vector, one may transformthe DNA library into a strain of S. cerevisiae with a specific mutationand select for the correction of the corresponding phenotype. Aftersub-cloning and sequencing the DNA fragment that is able to restore thewild-type phenotype, one may use this fragment to eliminate the activityof the gene product encoded by OCH1 in P. pastoris using in vivomutagenesis and/or recombination techniques well-known to those skilledin the art.

Alternatively, if the entire genomic sequence of a particular host cell,e.g., fungus, of interest is known, one may identify such genes simplyby searching publicly available DNA databases, which are available fromseveral sources, such as NCBI, Swissprot. For example, by searching agiven genomic sequence or database with sequences from a known 1,6mannosyltransferase gene (e.g., OCH1 from S. cerevisiae), one canidentify genes of high homology in such a host cell genome which may(but do not necessarily) encode proteins that have1,6-mannosyltransferase activity. Nucleic acid sequence homology aloneis not enough to prove, however, that one has identified and isolated ahomolog encoding an enzyme having the same activity. To date, forexample, no data exist to show that an OCH1 deletion in P. pastoriseliminates the crucial initiating 1,6-mannosyltransferase activity.(Martinet, 1998; WO 02/00856 A2). Thus, no data prove that the P.pastoris OCH1 gene homolog actually encodes that function. Thatdemonstration is provided for the first time herein.

Homologs to several S. cerevisiae mannosyltransferases have beenidentified in P. pastoris using these approaches. Homologous genes oftenhave similar functions to genes involved in the mannosylation ofproteins in S. cerevisiae and thus their deletion may be used tomanipulate the glycosylation pattern in P. pastoris or, by analogy, inany other host cell, e.g., fungus, plant, insect or animal cells, withsimilar glycosylation pathways.

The creation of gene knock-outs, once a given target gene sequence hasbeen determined, is a well-established technique in the art and can becarried out by one of ordinary skill in the art (see, e.g., Rothstein,1991). The choice of a host organism may be influenced by theavailability of good transformation and gene disruption techniques.

If several mannosyltransferases are to be knocked out, the methoddeveloped by Alani, 1987, for example, enables the repeated use of aselectable marker, e.g., the URA3 marker in yeast, to sequentiallyeliminate all undesirable endogenous mannosyltransferase activity. Thistechnique has been refined by others but basically involves the use oftwo repeated DNA sequences, flanking a counter selectable marker. Forexample: URA3 may be used as a marker to ensure the selection of atransformants that have integrated a construct. By flanking the URA3marker with direct repeats one may first select for transformants thathave integrated the construct and have thus disrupted the target gene.After isolation of the transformants, and their characterization, onemay counter select in a second round for those that are resistant to5-fluoroorotic acid (5-FOA). Colonies that are able to survive on platescontaining 5-FOA have lost the URA3 marker again through a crossoverevent involving the repeats mentioned earlier. This approach thus allowsfor the repeated use of the same marker and facilitates the disruptionof multiple genes without requiring additional markers. Similartechniques for sequential elimination of genes adapted for use inanother eukaryotic host cells with other selectable andcounter-selectable markers may also be used.

Eliminating specific mannosyltransferases, such as 1,6mannosyltransferase (OCH1) or mannosylphosphate transferases (MNN6, orgenes complementing lbd mutants) or regulators (MNN4) in P. pastorisenables one to create engineered strains of this organism whichsynthesize primarily Man₈GlcNAc₂ and which can be used to further modifythe glycosylation pattern to resemble more complex glycoform structures,e.g., those produced in mammalian, e.g., human cells. A preferredembodiment of this method utilizes DNA sequences encoding biochemicalglycosylation activities to eliminate similar or identical biochemicalfunctions in P. pastoris to modify the glycosylation structure ofglycoproteins produced in the genetically altered P. pastoris strain.

Methods used to engineer the glycosylation pathway in yeasts asexemplified herein can be used in filamentous fungi to produce apreferred substrate for subsequent modification. Strategies formodifying glycosylation pathways in A. niger and other filamentousfungi, for example, can be developed using protocols analogous to thosedescribed herein for engineering strains to produce human-likeglycoproteins in yeast. Undesired gene activities involved in 1,2mannosyltransferase activity, e.g., KTR/KRE homologs, are modified oreliminated. A filamentous fungus, such as Aspergillus, is a preferredhost because it lacks the 1,6 mannosyltransferase activity and as such,one would not expect a hypermannosylating gene activity, e.g. OCH1, inthis host. By contrast, other desired activities (e.g.,α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT),galactosyltransferase (GalT) and sialyltransferase (ST)) involved inglycosylation are introduced into the host using the targeting methodsof the invention.

Engineering or Selecting Hosts Having Diminished Initiating α-1,6Mannosyltransferase Activity

In a preferred embodiment, the method of the invention involves makingor using a host cell which is diminished or depleted in the activity ofan initiating α-1,6-mannosyltransferase, i.e., an initiation specificenzyme that initiates outer chain mannosylation on the α-1,3 arm of theMan₃GlcNAc₂ core structure. In S. cerevisiae, this enzyme is encoded bythe OCH1 gene. Disruption of the OCH1 gene in S. cerevisiae results in aphenotype in which N-linked sugars completely lack the polymannose outerchain. Previous approaches for obtaining mammalian-type glycosylation infungal strains have required inactivation of OCH1 (see, e.g., Chiba,1998). Disruption of the initiating α-1,6-mannosyltransferase activityin a host cell of the invention may be optional, however (depending onthe selected host cell), as the Och1p enzyme requires an intactMan₈GlcNAc₂ for efficient mannose outer chain initiation. Thus, hostcells selected or produced according to this invention which accumulateoligosaccharides having seven or fewer mannose residues may producehypoglycosylated N-glycans that will likely be poor substrates for Och1p(see, e.g., Nakayama, 1997).

The OCH1 gene was cloned from P. pastoris (Example 1) and K. lactis(Example 18), as described. The nucleic acid and amino acid sequences ofthe OCH1 gene from K. lactis are set forth in SEQ ID NOS: 41 and 42.Using gene-specific primers, a construct was made from each clone todelete the OCH1 gene from the genome of P. pastoris and K. lactis(Examples 1 and 18, respectively). Host cells depleted in initiatingα-1,6-mannosyltransferase activity and engineered to produce N-glycanshaving a Man₅GlcNAc₂ carbohydrate structure were thereby obtained (see,e.g., FIGS. 5 and 6; Examples 11 and 18).

Thus, in another embodiment, the invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofat least forty-five, preferably at least 50, more preferably at least 60and most preferably 75 or more nucleotide residues of the K. lactis OCH1gene (SEQ ID NO: 41), and homologs, variants and derivatives thereof.The invention also provides nucleic acid molecules that hybridize understringent conditions to the above-described nucleic acid molecules.Similarly, isolated polypeptides (including muteins, allelic variants,fragments, derivatives, and analogs) encoded by the nucleic acidmolecules of the invention are provided. Also provided are vectors,including expression vectors, which comprise the above nucleic acidmolecules of the invention, as described further herein. Similarly, hostcells transformed with the nucleic acid molecules or vectors of theinvention are provided.

Host Cells Enriched in Man₅GlcNAc₂

A preferred host cell of the invention is a lower eukaryotic cell, e.g.,yeast, a unicellular and multicellular or filamentous fungus. However, awide variety of host cells are envisioned as being useful in the methodsof the invention. Plant cells or insect cells, for instance, may beengineered to express a human-like glycoprotein according to theinvention (Examples 19 and 20). Likewise, a variety of non-human,mammalian host cells may be altered to express more human-like orotherwise altered glycoproteins using the methods of the invention. Asone of skill in the art will appreciate, any eukaryotic host cell(including a human cell) may be used in conjunction with a library ofthe invention to express one or more chimeric proteins which is targetedto a subcellular location, e.g., organelle, in the host cell where theactivity of the protein is modified, and preferably is enhanced. Such aprotein is preferably—but need not necessarily be—an enzyme involved inprotein glycosylation, as exemplified herein. It is envisioned that anyprotein coding sequence may be targeted and selected for modifiedactivity in a eukaryotic host cell using the methods described herein.

Lower eukaryotes that are able to produce glycoproteins having theattached N-glycan Man₅GlcNAc₂ are particularly useful because (a)lacking a high degree of mannosylation (e.g. greater than 8 mannoses perN-glycan, or especially 30-40 mannoses), they show reducedimmunogenicity in humans; and (b) the N-glycan is a substrate forfurther glycosylation reactions to form an even more human-likeglycoform, e.g., by the action of GlcNAc transferase I (FIG. 1B; β1,2GnTI) to form GlcNAcMan₅GlcNAc₂. A yield is obtained of greater than 30mole %, more preferably a yield of 50-100 mole %, glycoproteins withN-glycans having a Man₅GlcNAc₂ structure. In a preferred embodiment,more than 50% of the Man₅GlcNAc₂ structure is shown to be a substratefor a GnTI activity and can serve as such a substrate in vivo. Lowereukaryotes may also be useful host cells of the invention because theytypically lack (unless otherwise engineered to add) galactose, fucoseand N-acetylglycosamine. Thus, recombinant proteins lacking, e.g.,fucose, may be made in these host cells.

Preferred lower eukaryotes of the invention include but are not limitedto: any Pichia sp., including but limited to: Pichia pastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichiamethanolica; any Saccharomyces sp including but not limited to:Saccharomyces cerevisiae, Hansenula polymorpha; any Kluyveromyces sp.including but not limited to: Kluyveromyces lactis; Candida albicans;Aspergillus nidulans; Aspergillus niger; Aspergillus oryzae; Trichodermareseei; Chrysosporium lucknowense; any Fusarium sp. including but notlimited to: Fusarium gramineum and Fusarium venenatum; and Neurosporacrassa.

In each above embodiment, the method is directed to making a host cellin which the oligosaccharide precursors are enriched in Man₅GlcNAc₂.These structures are desirable because they may then be processed bytreatment in vitro, for example, using the method of U.S. Pat. No.5,834,251. In a preferred embodiment, however, precursors enriched inMan₅GlcNAc₂ are processed by at least one further glycosylation reactionin vivo—with glycosidases (e.g., α-mannosidases) andglycosyltransferases (e.g., GnTI)—to produce human-like N-glycans.Oligosaccharide precursors enriched in Man₅GlcNAc₂, for example, arepreferably processed to those having GlcNAcMan_(X)GlcNAc₂ corestructures, wherein X is 3, 4 or 5, and is preferably 3. N-glycanshaving a GlcNAcMan_(X)GlcNAc₂ core structure where X is greater than 3may be converted to GlcNAcMan₃GlcNAc₂, e.g., by treatment with an α-1,3and/or α-1,6 mannosidase activity, where applicable. Additionalprocessing of GlcNAcMan₃GlcNAc₂ by treatment with glycosyltransferases(e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ core structures which may thenbe modified, as desired, e.g., by ex vivo treatment or by heterologousexpression in the host cell of additional glycosylation enzymes,including glycosyltransferases, sugar transporters and mannosidases (seebelow), to comprise specific human-like N-glycans, as desired.

Preferred human-like glycoproteins which may be produced according tothe invention include those that comprise N-glycans having seven orfewer, or three or fewer, mannose residues; and that comprise one ormore sugars selected from the group consisting of galactose, GlcNAc,sialic acid, and fucose. Other preferred human-like glycoproteins whichmay be produced according to the invention lack fucose.

Formation of Complex N-Glycans

Formation of complex N-glycan synthesis is a sequential process by whichspecific sugar residues are removed and attached to the coreoligosaccharide structure. In higher eukaryotes, this is achieved byhaving the substrate sequentially exposed to various processing enzymes.These enzymes carry out specific reactions depending on their particularlocation within the entire processing cascade. This “assembly line”consists of ER, early, medial and late Golgi, and the trans Golginetwork all with their specific processing environment. To re-create theprocessing of human glycoproteins in the Golgi and ER of lowereukaryotes, numerous enzymes (e.g. glycosyltransferases, glycosidases,phosphatases and transporters) have to be expressed and specificallytargeted to these organelles, and preferably, in a location so that theyfunction most efficiently in relation to their environment as well as toother enzymes in the pathway.

Because one goal of the methods described herein is to achieve a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the hostcell chromosome involves careful planning. As described above, one ormore genes which encode enzymes known to be characteristic of non-humanglycosylation reactions are preferably deleted. The engineered cellstrain is transformed with a range of different genes encoding desiredactivities, and these genes are transformed in a stable fashion, therebyensuring that the desired activity is maintained throughout thefermentation process.

Any combination of the following enzyme activities may be engineeredsingly or multiply into the host using methods of the invention:sialyltransferases, mannosidases, fucosyltransferases,galactosyltransferases, GlcNAc transferases, ER and Golgi specifictransporters (e.g. syn- and antiport transporters for UDP-galactose andother precursors), other enzymes involved in the processing ofoligosaccharides, and enzymes involved in the synthesis of activatedoligosaccharide precursors such as UDP-galactose andCMP-N-acetylneuraminic acid. Preferably, enzyme activities areintroduced on one or more nucleic acid molecules (see also below).Nucleic acid molecules may be introduced singly or multiply, e.g., inthe context of a nucleic acid library such as a combinatorial library ofthe invention. It is to be understood, however, that single or multipleenzymatic activities may be introduced into a host cell in any fashion,including but not limited to protein delivery methods and/or by use ofone or more nucleic acid molecules without necessarily using a nucleicacid library or combinatorial library of the invention.

Expression of Glycosyltransferases to Produce Complex N-Glycans

With DNA sequence information, the skilled artisan can clone DNAmolecules encoding one or more GnT activities such as GnTI, II, III, IVor IV (e.g., Examples 3 and 4); galactosyltransferase activities(Example 4); and/or sialyltransferase activities (Examples 6 and 17).Using standard techniques well-known to those of skill in the art,nucleic acid molecules encoding one or more of the above describedenzymes (or encoding catalytically active fragments thereof) may beinserted into appropriate expression vectors under the transcriptionalcontrol of promoters and other expression control sequences capable ofdriving transcription in a selected host cell of the invention, e.g., afungal host such as Pichia sp., Kluyveromyces sp. and Aspergillus sp.,as described herein, such that one or more of these mammalian enzymesmay be actively expressed in a host cell of choice for production of ahuman-like complex glycoprotein (e.g., Examples 15, 16, 17, 19 and 20).

Several individual glycosyltransferases have been cloned and expressedin S. cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and otherfungi, without however demonstrating the desired outcome of“humanization” on the glycosylation pattern of the organisms (Yoshida,1995; Schwientek, 1995; Kalsner, 1995). It was speculated that thecarbohydrate structure required to accept sugars by the action of suchglycosyltransferases was not present in sufficient amounts, which mostlikely contributed to the lack of complex N-glycan formation.

A preferred method of the invention provides the functional expressionof a GnT, such as GnTI, in the early or medial Golgi apparatus as wellas ensuring a sufficient supply of UDP-GlcNAc (e.g., by expression of aUDP-GlcNAc transporter; see below).

Another preferred method of the invention provides functional expressionof a sialyltransferase, such as ST6Gal or ST3Gal, in the Golgi as wellas ensuring a sufficient supply of CMP-Sialic acid (e.g., by expressionof a CMP-Sialic acid biosynthetic pathway in the host as illustrated inExample 16). Sialyltransferases from many species are known and may beuseful in the present invention. Sialyltransferases are described forexample, in Harduin-Lepers, 2001; Hardeuin-Lepers, 2005; and Tsji, 1996.The disclosures of all of these references are hereby incorporatedherein by reference.

Methods for Providing Sugar Nucleotide Precursors to the Golgi Apparatus

For a glycosyltransferase to function satisfactorily in the Golgi, theenzyme requires a sufficient concentration of an appropriate nucleotidesugar, which is the high-energy donor of the sugar moiety added to anascent glycoprotein. In humans and other non-human eurkayotic cells,the full range of nucleotide sugar precursors (e.g.UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, UDP-galactose, etc.) are generallysynthesized in the cytosol and transported into the Golgi, where theyare attached to the core oligosaccharide by glycosyltransferases.

To replicate this process in host cells that do not comprise thesenucleotide precursors in the Golgi (e.g., in non-human host cells suchas lower eukaryotes), sugar nucleoside specific transporters have to beexpressed in the Golgi to ensure adequate levels of nucleoside sugarprecursors (Sommers, 1981; Sommers, 1982; Perez, 1987). Nucleotidesugars may be provided to the appropriate compartments, e.g., byexpressing in the host cell an exogenous gene encoding a sugarnucleotide transporter. The choice of transporter enzyme is influencedby the nature of the exogenous glycosyltransferase being used. Forexample, a GlcNAc transferase may require a UDP-GlcNAc transporter, afucosyltransferase may require a GDP-fucose transporter, agalactosyltransferase may require a UDP-galactose transporter, and asialyltransferase may require a CMP-sialic acid transporter.

The added transporter protein conveys a nucleotide sugar from thecytosol into the Golgi apparatus, where the nucleotide sugar may bereacted by the glycosyltransferase, e.g. to elongate an N-glycan. Thereaction liberates a nucleoside diphosphate or monophosphate, e.g. UDP,GDP, or CMP. Nucleoside monophosphates can be directly exported from theGolgi in exchange for nucleoside triphosphate sugars by an antiportmechanism. Accumulation of a nucleoside diphosphate, however, inhibitsthe further activity of a glycosyltransferase. As this reaction appearsto be important for efficient glycosylation, it is frequently desirableto provide an expressed copy of a gene encoding a nucleotidediphosphatase. The diphosphatase (specific for UDP or GDP asappropriate) hydrolyzes the diphosphonucleoside to yield a nucleosidemonosphosphate and inorganic phosphate.

Suitable transporter enzymes, which are typically of mammalian origin,are described below. Such enzymes may be engineered into a selected hostcell using the methods of the invention (see also Examples 7-10).

In another example, α 2,3- or α 2,6-sialyltransferase caps galactoseresidues with sialic acid in the trans-Golgi and TGN of humans leadingto a mature form of the glycoprotein (FIG. 1B). To reengineer thisprocessing step into a lower eukaryotic host cell and other host cellswhich naturally lack sialyltransferase activity will require (1) α 2,3-or α 2,6-sialyltransferase activity and (2) a sufficient supply ofCMP-N-acetyl neuraminic acid, in the late Golgi (Examples 6, 16 and 17).To obtain sufficient α 2,3-sialyltransferase activity in the secretorypathway (e.g. late Golgi), for example, the catalytic domain of a knownsialyltransferase (e.g. from humans) has to be directed to the secretorypathway in lower eukaryotic host cells (see above). Likewise,transporters have to be engineered to allow the transport ofCMP-N-acetyl neuraminic acid into the same location of the secretorypathway (e.g. late Golgi). There is currently no indication that hostcells such as lower eukaryotic host cells synthesize or can eventransport sufficient amounts of CMP-N-acetyl neuraminic acid into theGolgi. Consequently, to ensure the adequate supply of substrate for thecorresponding glycosyltransferases, one has to metabolically engineerthe production of CMP-sialic acid into these host cells.

UDP-N-Acetylglucosamine Transporter

The cDNA of human UDP-N-acetylglucosamine transporter, which wasrecognized through a homology search in the expressed sequence tagsdatabase (dbEST), has been cloned (Ishida, 1999). The mammalian Golgimembrane transporter for UDP-N-acetylglucosamine was cloned byphenotypic correction with cDNA from canine kidney cells (MDCK) of arecently characterized Kluyveromyces lactis mutant deficient in Golgitransport of the above nucleotide sugar (Guillen, 1998). Resultsdemonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has allof the necessary information for the protein to be expressed andtargeted functionally to the Golgi apparatus of yeast and that twoproteins with very different amino acid sequences may transport the samesolute within the same Golgi membrane (Guillen, 1998).

Accordingly, one may incorporate the expression of a UDP-GlcNActransporter in a host cell by means of a nucleic acid construct whichmay contain, for example: (1) a region by which the transformedconstruct is maintained in the cell (e.g. origin of replication or aregion that mediates chromosomal integration), (2) a marker gene thatallows for the selection of cells that have been transformed, includingcounterselectable and recyclable markers such as ura3 or T-urf13(Soderholm, 2001) or other well characterized selection-markers (e.g.,his4, bla, Sh ble etc.), (3) a gene or fragment thereof encoding afunctional UDP-GlcNAc transporter, e.g. from K. lactis (Abeijon, 1996),or from H. sapiens (Ishida, 1996), and (4) a promoter activating theexpression of the above mentioned localization/catalytic domain fusionconstruct library.

GDP-Fucose Transporter

The rat liver Golgi membrane GDP-fucose transporter has been identifiedand purified by Puglielli, L. and C. B. Hirschberg (Puglielli, 1999).The corresponding gene has not been identified; however, N-terminalsequencing can be used for the design of oligonucleotide probes specificfor the corresponding gene. These oligonucleotides can be used as probesto clone the gene encoding for GDP-fucose transporter.

UDP-Galactose Transporter

Two heterologous genes, gmal2(+) encoding alpha1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomycespombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter,have been functionally expressed in S. cerevisiae to examine theintracellular conditions required for galactosylation. Correlationbetween protein galactosylation and UDP-galactose transport activityindicated that an exogenous supply of UDP-Gal transporter, rather thanalpha 1,2 GalT played a key role for efficient galactosylation in S.cerevisiae (Kainuma, 1999). Likewise, an UDP-galactose transporter fromS. pombe was cloned (Aoki, 1999; Segawa, 1999).

CMP-N-Acetylneuraminic Acid (CMP-Sialic Acid) Transporter

Human CMP-sialic acid transporter (hCST) has been cloned and expressedin Lec 8 CHO cells (Aoki, 1999; Eckhardt, 1997). The functionalexpression of the murine CMP-sialic acid transporter was achieved inSaccharomyces cerevisiae (Berninsone, 1997). Sialic acid has been foundin some fungi, however it is not clear whether the chosen host systemwill be able to supply sufficient levels of CMP-Sialic acid. Sialic acidcan be either supplied in the medium or alternatively fungal pathwaysinvolved in sialic acid synthesis can also be integrated into the hostgenome as described below.

Expression of Diphosphatases

When sugars are transferred onto a glycoprotein, either a nucleosidediphosphate or monophosphate is released from the sugar nucleotideprecursors. While monophosphates can be directly exported in exchangefor nucleoside triphosphate sugars by an antiport mechanism,diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g.GDPase) to yield nucleoside monophosphates and inorganic phosphate priorto being exported. This reaction appears to be important for efficientglycosylation, as GDPase from S. cerevisiae has been found to benecessary for mannosylation. However, the enzyme only has 10% of theactivity towards UDP (Berninsone, 1994). Lower eukaryotes often do nothave UDP-specific diphosphatase activity in the Golgi as they do notutilize UDP-sugar precursors for glycoprotein synthesis in the Golgi.Schizosaccharomyces pombe, a yeast which adds galactose residues to cellwall polysaccharides (from UDP-galactose), was found to have specificUDPase activity, further suggesting the requirement for such an enzyme(Berninsone, 1994). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productis important to prevent glycosyltransferase inhibition in the lumen ofthe Golgi (Khatara, 1974).

Formation of Hybrid Glycoproteins

With minor modifications, the same “assembly line” of enzymes andglycosylation pathway may be utilized to accomplish the formation ofcomplex N-glycans. The primary difference is that after action of GNT Ion the Man5GlcNAc2 core to produce GlcNAcMan5GlcNAc2, it is notnecessary that the GlcNAcMan5GlcNAc2 be exposed to the actionmannosidase II to form GlcNAcMan3GlcNAc2. See, e.g., Gerngross,WO02/00879 and U.S. Pat. No. 7,029,872.

Methods for Altering N-Glycans in a Host by Expressing a TargetedEnzymatic Activity from a Nucleic Acid Molecule

In one preferred embodiment, the invention provides a method forproducing a glycoprotein comprising Man₅GlcNAc₂. In one preferredembodiment, a nucleic acid molecule encoding one or more mannosidaseactivities involved in the production of Man₅GlcNAc₂ from Man₈GlcNAc₂ orMan₉GlcNAc₂ is introduced into the host.

In another embodiment, the invention provides a method for producing ahuman-like glycoprotein in a non-human eukaryotic host cell comprisingthe step of introducing into the host cell one or more nucleic acidmolecules that encode an enzyme or enzymes for production ofNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ carbohydrate structure.

The invention additionally relates to methods for making alteredglycoproteins in a host cell comprising the step of introducing into thehost cell a nucleic acid molecule which encodes one or moreglycosylation enzymes or activities. Preferred enzyme activities areselected from the group consisting of UDP-GlcNAc transferase,UDP-galactosyltransferase, GDP-fucosyltransferase,CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactosetransporter, GDP-fucose transporter, CMP-sialic acid transporter, andnucleotide diphosphatases. In a particularly preferred embodiment, thehost is selected or engineered to express two or more enzymaticactivities in which the product of one activity increases substratelevels of another activity, e.g., a glycosyltransferase and acorresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporteractivities. In another preferred embodiment, the host is selected orengineered to expresses an activity to remove products which may inhibitsubsequent glycosylation reactions, e.g. a UDP- or GDP-specificdiphosphatase activity.

Preferred methods of the invention involve expressing one or moreenzymatic activities from a nucleic acid molecule in a host cell andcomprise the step of targeting at least one enzymatic activity to adesired subcellular location (e.g., an organelle) by forming a fusionprotein comprising a catalytic domain of the enzyme and a cellulartargeting signal peptide, e.g., a heterologous signal peptide which isnot normally ligated to or associated with the catalytic domain. Thefusion protein is encoded by at least one genetic construct (“fusionconstruct”) comprising a nucleic acid fragment encoding a cellulartargeting signal peptide ligated in the same translational reading frame(“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g.,glycosylation enzyme), or catalytically active fragment thereof.

The targeting signal peptide component of the fusion construct orprotein is preferably derived from a member of the group consisting of:membrane-bound proteins of the ER or Golgi, retrieval signals, Type IImembrane proteins, Type I membrane proteins, membrane spanningnucleotide sugar transporters, mannosidases, sialyltransferases,glucosidases, mannosyltransferases and phosphomannosyltransferases.

The catalytic domain component of the fusion construct or protein ispreferably derived from a glycosidase, mannosidase or aglycosyltransferase activity derived from a member of the groupconsisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GalT,Fucosyltransferase and Sialyltransferase. The catalytic domainpreferably has a pH optimum within 1.4 pH units of the average pHoptimum of other representative enzymes in the organelle in which theenzyme is localized, or has optimal activity at a pH between 5.1 and8.0.

Selecting a Glycosylation Enzyme: pH Optima and Subcellular Localization

In one embodiment of the invention, a human-like glycoprotein is madeefficiently in a non-human eukaryotic host cell by introducing into asubcellular compartment of the cell a glycosylation enzyme selected tohave a pH optimum similar to the pH optima of other enzymes in thetargeted subcellular compartment. For example, most enzymes that areactive in the ER and Golgi apparatus of S. cerevisiae have pH optimathat are between about 6.5 and 7.5 (see Table 3). Because theglycosylation of proteins is a highly evolved and efficient process, theinternal pH of the ER and the Golgi is likely also in the range of about6-8. All previous approaches to reduce mannosylation by the action ofrecombinant mannosidases in fungal hosts, however, have introducedenzymes that have a pH optimum of around pH 5.0 (Martinet, 1998 andChiba, 1998). At pH 7.0, the in vitro determined activity of thosemannosidases is reduced to less than 10%, which is likely insufficientactivity at their point of use, namely, the ER and early Golgi, for theefficient in vivo production of Man₅GlcNAc₂ on N-glycans.

Accordingly, a preferred embodiment of this invention targets a selectedglycosylation enzyme (or catalytic domain thereof), e.g., anα-mannosidase, to a subcellular location in the host cell (e.g., anorganelle) where the pH optimum of the enzyme or domain is within 1.4 pHunits of the average pH optimum of other representative marker enzymeslocalized in the same organelle(s). The pH optimum of the enzyme to betargeted to a specific organelle should be matched with the pH optimumof other enzymes found in the same organelle to maximize the activityper unit enzyme obtained. Table 3 summarizes the activity ofmannosidases from various sources and their respective pH optima. Table4 summarizes their typical subcellular locations.

TABLE 3 Mannosidases And Their pH Optimum. pH Source Enzyme optimumReference Aspergillus saitoi α-1,2-mannosidase 5.0 Ichishima, 1999Trichoderma α-1,2-mannosidase 5.0 Maras, 2000 reesei Penicilliumα-D-1,2- 5.0 Yoshida, 1993 citrinum mannosidase C.elegansα-1,2-mannosidase 5.5 see FIG. 11 Aspergillus α-1,2-mannosidase 6.0Eades and Hintz, 2000 nidulans Homo sapiens α-1,2-mannosidase 6.0IA(Golgi) Homo sapiens IB α-1,2-mannosidase 6.0 (Golgi) LepidopteranType I α-1,2-Man₆- 6.0 Ren, 1995 insect cells mannosidase Homo sapiensα-D-mannosidase 6.0 Chandrasekaran, 1984 Xanthomonas α-1,2,3-mannosidase6.0 U.S. Pat. No. 6,300,113 manihotis Mouse IB (Golgi) α-1,2-mannosidase6.5 Schneikert, 1994 Bacillus sp. α-D-1,2- 7.0 Maruyama, 1994 (secreted)mannosidase

In a preferred embodiment, a particular enzyme or catalytic domain istargeted to a subcellular location in the host cell by means of achimeric fusion construct encoding a protein comprising a cellulartargeting signal peptide not normally associated with the enzymaticdomain. Preferably, an enzyme or domain is targeted to the ER, theearly, medial or late Golgi of the trans Golgi apparatus of the hostcell.

In a more preferred embodiment, the targeted glycosylation enzyme is aglycosyltransferase or a glycosidase (such as a mannosidase). In anespecially preferred embodiment, an enzyme having sialyltransferaseactivity is targeted to the late Golgi, where the early reactions ofglycosylation occur. While the methods disclosed herein are useful forproducing a human-like glycoprotein in a non-human host cell, it will beappreciated that the methods discussed herein are also useful moregenerally for modifying carbohydrate profiles of a glycoprotein in anyeukaryotic host cell, including human host cells. See Gerngross,WO02/00879.

Targeting sequences which mediate retention of proteins in certainorganelles of the host cell secretory pathway are well-known anddescribed in the scientific literature and public databases, asdiscussed in more detail below with respect to libraries for selectionof targeting sequences and targeted enzymes. Such subcellular targetingsequences may be used alone or in combination to target a selectedglycosylation enzyme (or catalytic domain thereof) to a particularsubcellular location in a host cell, i.e., especially to one where theenzyme will have enhanced or optimal activity based on pH optima or thepresence of other stimulatory factors.

When one attempts to trim high mannose structures to yield Man₅GlcNAc₂in the ER or the Golgi apparatus of a host cell such as S. cerevisiae,for example, one may choose any enzyme or combination of enzymes that(1) has a sufficiently close pH optimum (i.e. between pH 5.2 and pH7.8), and (2) is known to generate, alone or in concert, the specificisomeric Man₅GlcNAc₂ structure required to accept subsequent addition ofGlcNAc by GnTI. Any enzyme or combination of enzymes that is shown togenerate a structure that can be converted to GlcNAcMan₅GlcNAc₂ by GnTIin vitro would constitute an appropriate choice. This knowledge may beobtained from the scientific literature or experimentally.

For example, one may determine whether a potential mannosidase canconvert Man₈GlcNAc₂-2AB (2-aminobenzamide) to Man₅GlcNAc₂-AB and thenverify that the obtained Man₅GlcNAc₂-2AB structure can serve a substratefor GnTI and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro. MannosidaseIA from a human or murine source, for example, would be an appropriatechoice (see, e.g., Example 11). Examples described herein utilize2-aminobenzamide labeled N-linked oligomannose followed by HPLC analysisto make this determination.

TABLE 4 Cellular Location And pH Optima Of Various Glycosylation-RelatedEnzymes Of S.cerevisiae. Gene Activity Location pH optimum Reference(s)KTR1 α-1,2 Golgi 7.0 Romero, 1997 mannosyltransferase MNS1 α-1,2-mannosidase ER 6.5 CWH41 glucosidase I ER 6.8 — mannosyltransferaseGolgi 7-8 Lehele, 1974 KRE2 α-1,2 Golgi 6.5-9.0 Romero, 1997mannosyltransferase

Accordingly, a glycosylation enzyme such as an α-1,2-mannosidase enzymeused according to the invention has an optimal activity at a pH ofbetween 5.1 and 8.0. In a preferred embodiment, the enzyme has anoptimal activity at a pH of between 5.5 and 7.5. The C. elegansmannosidase enzyme, for example, works well in the methods of theinvention and has an apparent pH optimum of about 5.5).

The experiment which illustrates the pH optimum for an α-1,2-mannosidaseenzyme is described in Example 14. A chimeric fusion protein BB27-2(Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31), which leaksinto the medium was subjected to various pH ranges to determine theoptimal activity of the enzyme. The results of the experiment show thatthe α-1,2-mannosidase has an optimal pH of about 5.5 for its function(FIG. 11).

In a preferred embodiment, a single cloned mannosidase gene is expressedin the host organism. However, in some cases it may be desirable toexpress several different mannosidase genes, or several copies of oneparticular gene, in order to achieve adequate production of Man₅GlcNAc₂.In cases where multiple genes are used, the encoded mannosidasespreferably all have pH optima within the preferred range of about 5.1 toabout 8.0, or especially between about 5.5 and about 7.5. Preferredmannosidase activities include α-1,2-mannosidases derived from mouse,human, Lepidoptera, Aspergillus nidulans, or Bacillus sp., C. elegans,D. melanogaster, P. citrinum, X. laevis or A. nidulans.

In Vivo Alteration of Host Cell Glycosylation Using a Combinatorial DNALibrary

Certain methods of the invention are preferably (but need notnecessarily be) carried out using one or more nucleic acid libraries. Anexemplary feature of a combinatorial nucleic acid library of theinvention is that it comprises sequences encoding cellular targetingsignal peptides and sequences encoding proteins to be targeted (e.g.,enzymes or catalytic domains thereof, including but not limited to thosewhich mediate glycosylation).

In one embodiment, a combinatorial nucleic acid library comprises: (a)at least two nucleic acid sequences encoding different cellulartargeting signal peptides; and (b) at least one nucleic acid sequenceencoding a polypeptide to be targeted. In another embodiment, acombinatorial nucleic acid library comprises: (a) at least one nucleicacid sequence encoding a cellular targeting signal peptide; and (b) atleast two nucleic acid sequences encoding a polypeptide to be targetedinto a host cell. As described further below, a nucleic acid sequencederived from (a) and a nucleic acid sequence derived from (b) areligated to produce one or more fusion constructs encoding a cellulartargeting signal peptide functionally linked to a polypeptide domain ofinterest. One example of a functional linkage is when the cellulartargeting signal peptide is ligated to the polypeptide domain ofinterest in the same translational reading frame (“in-frame”).

In a preferred embodiment, a combinatorial DNA library expresses one ormore fusion proteins comprising cellular targeting signal peptidesligated in-frame to catalytic enzyme domains. The encoded fusion proteinpreferably comprises a catalytic domain of an enzyme involved inmammalian- or human-like modification of N-glycans. In a more preferredembodiment, the catalytic domain is derived from an enzyme selected fromthe group consisting of mannosidases, glycosyltransferases and otherglycosidases which is ligated in-frame to one or more targeting signalpeptides. The enzyme domain may be exogenous and/or endogenous to thehost cell. A particularly preferred signal peptide is one normallyassociated with a protein that undergoes ER to Golgi transport.

The combinatorial DNA library of the present invention may be used forproducing and localizing in vivo enzymes involved in mammalian- orhuman-like N-glycan modification. The fusion constructs of thecombinatorial DNA library are engineered so that the encoded enzymes arelocalized in the ER, Golgi or the trans-Golgi network of the host cellwhere they are involved in producing particular N-glycans on aglycoprotein of interest. Localization of N-glycan modifying enzymes ofthe present invention is achieved through an anchoring mechanism orthrough protein-protein interaction where the localization peptideconstructed from the combinatorial DNA library localizes to a desiredorganelle of the secretory pathway such as the ER, Golgi or the transGolgi network.

An example of a useful N-glycan, which is produced efficiently and insufficient quantities for further modification by human-like (complex)glycosylation reactions is Man₅GlcNAc₂. A sufficient amount ofMan₅GlcNAc₂ is needed on a glycoprotein of interest for furtherhuman-like processing in vivo (e.g., more than 30 mole %). TheMan₅GlcNAc₂ intermediate may be used as a substrate for further N-glycanmodification to produce GlcNAcMan₅GlcNAc₂ (FIG. 1B; see above).Accordingly, the combinatorial DNA library of the present invention maybe used to produce enzymes which subsequently produce GlcNAcMan₅GlcNAc₂,or other desired complex N-glycans, in a useful quantity.

A further aspect of the fusion constructs produced using thecombinatorial DNA library of the present invention is that they enablesufficient and often near complete intracellular N-glycan trimmingactivity in the engineered host cell. Preferred fusion constructsproduced by the combinatorial DNA library of the invention encode aglycosylation enzyme, e.g., a mannosidase, which is effectivelylocalized to an intracellular host cell compartment and thereby exhibitsvery little and preferably no extracellular activity. The preferredfusion constructs of the present invention that encode a mannosidaseenzyme are shown to localize where the N-glycans are modified, namely,the ER and the Golgi. The fusion enzymes of the present invention aretargeted to such particular organelles in the secretory pathway wherethey localize and act upon N-glycans such as Man₈GlcNAc₂ to produceMan₅GlcNAc₂ on a glycoprotein of interest.

Enzymes produced by the combinatorial DNA library of the presentinvention can modify N-glycans on a glycoprotein of interest as shownfor K3 or IFN-β proteins expressed in P. pastoris, as shown in FIG. 5and FIG. 6, respectively (see also Examples 2 and 11). It is, however,appreciated that other types of glycoproteins, without limitation,including erythropoietin, cytokines such as interferon-α, interferon-β,interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factorssuch as factor VIII, factor IX, and human protein C, soluble IgEreceptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase,chymase, and urea trypsin inhibitor, IGF-binding protein, epidermalgrowth factor, growth hormone-releasing factor, annexin V fusionprotein, angiostatin, vascular endothelial growth factor-2, myeloidprogenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNaseII, α-feto proteins, AAT, rhTBP-1 (onercept, aka TNF Binding protein 1),TACI-Ig (transmembrane activator and calcium modulator and cyclophilinligand interactor), FSH (follicle stimulating hormone), GM-CSF, GLP-1 w/and w/o FC (glucagon like protein 1) IL-1 receptor agonist, sTNFr(enbrel, aka soluble TNF receptor Fc fusion) ATIII, rhThrombin,glucocerebrosidase and CTLA4-Ig (Cytotoxic T Lymphocyte associatedAntigen 4-Ig) may be glycosylated in this way.

Constructing a Combinatorial DNA Library of Fusion Constructs

A combinatorial DNA library of fusion constructs features one or morecellular targeting signal peptides (“targeting peptides”) generallyderived from N-terminal domains of native proteins (e.g., by makingC-terminal deletions). Some targeting peptides, however, are derivedfrom the C-terminus of native proteins (e.g. SEC12). Membrane-boundproteins of the ER or the Golgi are preferably used as a source fortargeting peptide sequences. These proteins have sequences encoding acytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr)which are varied in length. These regions are recognizable by proteinsequence alignments and comparisons with known homologs and/or otherlocalized proteins (e.g., comparing hydrophobicity plots).

The targeting peptides are indicated herein as short (s), medium (m) andlong (l) relative to the parts of a type II membrane. The targetingpeptide sequence indicated as short (s) corresponds to the transmembranedomain (tmd) of the membrane-bound protein. The targeting peptidesequence indicated as long (l) corresponds to the length of thetransmembrane domain (tmd) and the stem region (sr). The targetingpeptide sequence indicated as medium (m) corresponds to thetransmembrane domain (tmd) and approximately half the length of the stemregion (sr). The catalytic domain regions are indicated herein by thenumber of nucleotide deletion with respect to its wild-typeglycosylation enzyme.

Sub-Libraries

In some cases a combinatorial nucleic acid library of the invention maybe assembled directly from existing or wild-type genes. In a preferredembodiment, the DNA library is assembled from the fusion of two or moresub-libraries. By the in-frame ligation of the sub-libraries, it ispossible to create a large number of novel genetic constructs encodinguseful targeted protein domains such as those which have glycosylationactivities.

Catalytic Domain Sub-Libraries Encoding Glycosylation Activities

One useful sub-library includes DNA sequences encoding enzymes such asglycosidases (e.g., mannosidases), glycosyltransferases (e.g.,fucosyl-transferases, galactosyltransferases, glucosyltransferases),GlcNAc transferases and sialyltransferases. Catalytic domains may beselected from the host to be engineered, as well as from other relatedor unrelated organisms. Mammalian, plant, insect, reptile, algal orfungal enzymes are all useful and should be chosen to represent a broadspectrum of biochemical properties with respect to temperature and pHoptima. In a preferred embodiment, genes are truncated to give fragmentssome of which encode the catalytic domains of the enzymes. By removingendogenous targeting sequences, the enzymes may then be redirected andexpressed in other cellular loci.

The choice of such catalytic domains may be guided by the knowledge ofthe particular environment in which the catalytic domain is subsequentlyto be active. For example, if a particular glycosylation enzyme is to beactive in the late Golgi, and all known enzymes of the host organism inthe late Golgi have a certain pH optimum, or the late Golgi is known tohave a particular pH, then a catalytic domain is chosen which exhibitsadequate, and preferably maximum, activity at that pH, as discussedabove.

Targeting Peptide Sequence Sub-Libraries

Another useful sub-library includes nucleic acid sequences encodingtargeting signal peptides that result in localization of a protein to aparticular location within the ER, Golgi, or trans Golgi network. Thesetargeting peptides may be selected from the host organism to beengineered as well as from other related or unrelated organisms.Generally such sequences fall into three categories: (1) N-terminalsequences encoding a cytosolic tail (ct), a transmembrane domain (tmd)and part or all of a stem region (sr), which together or individuallyanchor proteins to the inner (lumenal) membrane of the Golgi; (2)retrieval signals which are generally found at the C-terminus such asthe HDEL (SEQ ID NO: 5) or KDEL (SEQ ID NO: 6) tetrapeptide; and (3)membrane spanning regions from various proteins, e.g., nucleotide sugartransporters, which are known to localize in the Golgi.

In the first case, where the targeting peptide consists of variouselements (ct, tmd and sr), the library is designed such that the ct, thetmd and various parts of the stem region are represented. Accordingly, apreferred embodiment of the sub-library of targeting peptide sequencesincludes ct, tmd, and/or sr sequences from membrane-bound proteins ofthe ER or Golgi. In some cases it may be desirable to provide thesub-library with varying lengths of sr sequence. This may beaccomplished by PCR using primers that bind to the 5′ end of the DNAencoding the cytosolic region and employing a series of opposing primersthat bind to various parts of the stem region.

Still other useful sources of targeting peptide sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL (SEQ ID NO: 5) orKDEL (SEQ ID NO: 6), which are typically found at the C-terminus ofproteins that are transported retrograde into the ER or Golgi. Stillother sources of targeting peptide sequences include (a) type IImembrane proteins, (b) the enzymes listed in Table 3, (c) membranespanning nucleotide sugar transporters that are localized in the Golgi,and (d) sequences referenced in Table 5. (The HDEL signal in column 1,cell 8 is shown in SEQ ID NO: 5).

TABLE 5 Sources Of Useful Compartmental Targeting Sequences Gene orLocation of Gene Sequence Organism Function Product MNSI A.nidulansα-1,2-mannosidase ER MNSI A.niger α-1,2-mannosidase ER MNSI S.cerevisiaeα-1,2-mannosidase ER GLSI S.cerevisiae glucosidase ER GLSI A.nigerglucosidase ER GLSI A.nidulans glucosidase ER HDEL Universal inretrieval signal ER at C- fungi terminus SEC12 S.cerevisiae COPIIvesicle protein ER/Golgi SEC12 A.niger COPII vesicle protein ER/GolgiOCH1 S.cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P.pastoris1,6-mannosyltransferase Golgi (cis) MNN9 S.cerevisiae1,6-mannosyltransferase Golgi complex MNN9 A.niger undetermined GolgiVAN1 S.cerevisiae undetermined Golgi VAN1 A.niger undetermined GolgiANP1 S.cerevisiae undetermined Golgi HOCI S.cerevisiae undeterminedGolgi MNN10 S.cerevisiae undetermined Golgi MNN10 A.niger undeterminedGolgi MNN11 S.cerevisiae undetermined Golgi (cis) MNN11 A.nigerundetermined Golgi (cis) MNT1 S.cerevisiae 1,2-mannosyltransferase Golgi(cis, medial KTR1 P.pastoris undetermined Golgi (medial) KRE2 P.pastorisUndetermined Golgi (medial) KTR3 P.pastoris Undetermined Golgi (medial)MNN2 S.cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1S.cerevisiae Undetermined Golgi (medial) KTR2 S.cerevisiae UndeterminedGolgi (medial) MNN1 S.cerevisiae 1,3-mannosyltransferase Golgi (trans)MNN6 S.cerevisiae Phosphomannosyltransferase Golgi (trans) 2,6 ST H.sapiens 2,6-sialyltransferase trans Golgi network UDP-Gal T S. pombeUDP-Gal transporter Golgi

In any case, it is highly preferred that targeting peptide sequences areselected which are appropriate for the particular enzymatic activity oractivities to function optimally within the sequence of desiredglycosylation reactions. For example, in developing a modifiedmicroorganism capable of terminal sialylation of nascent N-glycans, aprocess which occurs in the late Golgi in humans, it is desirable toutilize a sub-library of targeting peptide sequences derived from lateGolgi proteins. Similarly, the trimming of Man₈GlcNAc₂ by anα-1,2-mannosidase to give Man₅GlcNAc₂ is an early step in complexN-glycan formation in humans (FIG. 1B). It is therefore desirable tohave this reaction occur in the ER or early Golgi of an engineered hostmicroorganism. A sub-library encoding ER and early Golgi retentionsignals is used.

A series of fusion protein constructs (i.e., a combinatorial DNAlibrary) is then constructed by functionally linking one or a series oftargeting peptide sequences to one or a series of sequences encodingcatalytic domains. In a preferred embodiment, this is accomplished bythe in-frame ligation of a sub-library comprising DNA encoding targetingpeptide sequences (above) with a sub-library comprising DNA encodingglycosylation enzymes or catalytically active fragments thereof (seebelow).

The resulting library comprises synthetic genes encoding targetingpeptide sequence-containing fusion proteins. In some cases it isdesirable to provide a targeting peptide sequence at the N-terminus of afusion protein, or in other cases at the C-terminus. In some cases,targeting peptide sequences may be inserted within the open readingframe of an enzyme, provided the protein structure of individual foldeddomains is not disrupted. Each type of fusion protein is constructed (ina step-wise directed or semi-random fashion) and optimal constructs maybe selected upon transformation of host cells and characterization ofglycosylation patterns in transformed cells using methods of theinvention. Using such methods, lower eukaryotic host cells may beengineered to produce recombinant glycoprotein having desirable N-glucanproperties, e.g., tailored to the particular protein of interest.

Generating Additional Sequence Diversity

The method of this embodiment is most effective when a nucleic acid,e.g., a DNA library transformed into the host contains a large diversityof sequences, thereby increasing the probability that at least onetransformant will exhibit the desired phenotype. Single amino acidmutations, for example, may drastically alter the activity ofglycoprotein processing enzymes (Romero et al., 2000). Accordingly,prior to transformation, a DNA library or a constituent sub-library maybe subjected to one or more techniques to generate additional sequencediversity. For example, one or more rounds of gene shuffling, errorprone PCR, in vitro mutagenesis or other methods for generating sequencediversity, may be performed to obtain a larger diversity of sequenceswithin the pool of fusion constructs.

Expression Control Sequences

In addition to the open reading frame sequences described above, it isgenerally preferable to provide each library construct with expressioncontrol sequences, such as promoters, transcription terminators,enhancers, ribosome binding sites, and other functional sequences as maybe necessary to ensure effective transcription and translation of thefusion proteins upon transformation of fusion constructs into the hostorganism.

Suitable vector components, e.g., selectable markers, expression controlsequences (e.g., promoter, enhancers, terminators and the like) and,optionally, sequences required for autonomous replication in a hostcell, are selected as a function of which particular host cell ischosen. Selection criteria for suitable vector components for use in aparticular mammalian or a lower eukaryotic host cell are routine.Preferred lower eukaryotic host cells of the invention include but arenot limited to: any Pichia sp., including but limited to: Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichiamethanolica; any Saccharomyces sp including but not limited toSaccharomyces cerevisiae; Hansenula polymorpha; any Kluyveromyces sp.including but not limited to Kluyveromyces lactis; Candida albicans;Aspergillus nidulans; Aspergillus niger; Aspergillus oryzae; Trichodermareseei; Chrysosporium lucknowense; any Fusarium sp. including but notlimited to: Fusarium gramineum and Fusarium venenatum; and Neurosporacrassa. Where the host is Pichia pastoris, suitable promoters include,for example, the AOX1, AOX2, GAPDH and P40 promoters.

Selectable Markers

It is also preferable to provide each construct with at least oneselectable marker, such as a gene to impart drug resistance or tocomplement a host metabolic lesion. The presence of the marker is usefulin the subsequent selection of transformants; for example, in yeast theURA3, HIS4, SUC2, G418, BLA, or SH BLE genes may be used. A multitude ofselectable markers are known and available for use in yeast, fungi,plant, insect, mammalian and other eukaryotic host cells. A method fortransforming yeast cells by inactivating alternately at least twobiosynthetic pathways in methylotrophic yeast is described inUS2004/0229306, US2005/0170452, and Nett, 2005, which are herebyincorporated herein by reference. The method involves (a) inactivating afirst yeast gene in a pathway involved in synthesizing an amino acid ora nucleotide selected from the group consisting of adenine, arginine,histidine, lysine, methionine, proline and uracil with a firstselectable marker thereby rendering the host auxotrophic for the aminoacid or nucleotide; and then (b) inactivating a second yeast gene notfrom the same pathway that was inactivated in (a) involved insynthesizing an amino acid or a nucleotide selected from the groupconsisting of adenine, arginine, histidine, lysine, methionine, prolineand uracil using the yeast gene that was inactivated in (a) as a secondselectable marker.

Transformation

The nucleic acid library is then transformed into the host organism. Inyeast, any convenient method of DNA transfer may be used, such aselectroporation, the lithium chloride method, or the spheroplast method.In filamentous fungi and plant cells, conventional methods includeparticle bombardment, electroporation and agrobacterium mediatedtransformation. To produce a stable strain suitable for high-densityculture (e.g., fermentation in yeast), it is desirable to integrate theDNA library constructs into the host chromosome. In a preferredembodiment, integration occurs via homologous recombination, usingtechniques well-known in the art. For example, DNA library elements areprovided with flanking sequences homologous to sequences of the hostorganism. In this manner, integration occurs at a defined site in thehost genome, without disruption of desirable or essential genes.

In an especially preferred embodiment, library DNA is integrated intothe site of an undesired gene in a host chromosome, effecting thedisruption or deletion of the gene. For example, integration into thesites of the OCH1, MNN1, or MNN4 genes allows the expression of thedesired library DNA while preventing the expression of enzymes involvedin yeast hypermannosylation of glycoproteins. In other embodiments,library DNA may be introduced into the host via a nucleic acid molecule,plasmid, vector (e.g., viral or retroviral vector), chromosome, and maybe introduced as an autonomous nucleic acid molecule or by homologous orrandom integration into the host genome. In any case, it is generallydesirable to include with each library DNA construct at least oneselectable marker gene to allow ready selection of host organisms thathave been stably transformed. Recyclable marker genes such as ura3,which can be selected for or against, are especially suitable.

Screening And Selection Processes

After transformation of the host strain with the DNA library,transformants displaying a desired glycosylation phenotype are selected.Selection may be performed in a single step or by a series of phenotypicenrichment and/or depletion steps using any of a variety of assays ordetection methods. Phenotypic characterization may be carried outmanually or using automated high-throughput screening equipment.Commonly, a host microorganism displays protein N-glycans on the cellsurface, where various glycoproteins are localized.

One may screen for those cells that have the highest concentration ofterminal GlcNAc on the cell surface, for example, or for those cellswhich secrete the protein with the highest terminal GlcNAc content. Sucha screen may be based on a visual method, like a staining procedure, theability to bind specific terminal GlcNAc binding antibodies or lectinsconjugated to a marker (such lectins are available from E. Y.Laboratories Inc., San Mateo, Calif.), the reduced ability of specificlectins to bind to terminal mannose residues, the ability to incorporatea radioactively labeled sugar in vitro, altered binding to dyes orcharged surfaces, or may be accomplished by using a FluorescenceAssisted Cell Sorting (FACS) device in conjunction with a fluorophorelabeled lectin or antibody (Guillen, 1998).

Accordingly, intact cells may be screened for a desired glycosylationphenotype by exposing the cells to a lectin or antibody that bindsspecifically to the desired N-glycan. A wide variety ofoligosaccharide-specific lectins are available commercially (e.g., fromEY Laboratories, San Mateo, Calif.). Alternatively, antibodies tospecific human or animal N-glycans are available commercially or may beproduced using standard techniques. An appropriate lectin or antibodymay be conjugated to a reporter molecule, such as a chromophore,fluorophore, radioisotope, or an enzyme having a chromogenic substrate(Guillen, 1998).

Screening may then be performed using analytical methods such asspectrophotometry, fluorimetry, fluorescence activated cell sorting, orscintillation counting. In other cases, it may be necessary to analyzeisolated glycoproteins or N-glycans from transformed cells. Proteinisolation may be carried out by techniques known in the art. In apreferred embodiment, a reporter protein is secreted into the medium andpurified by affinity chromatography (e.g. Ni-affinity orglutathione-S-transferase affinity chromatography). In cases where anisolated N-glycan is preferred, an enzyme such asendo-β-N-acetylglucosaminidase (Genzyme Co., Boston, Mass.; New EnglandBiolabs, Beverly, Mass.) may be used to cleave the N-glycans fromglycoproteins. Isolated proteins or N-glycans may then be analyzed byliquid chromatography (e.g. HPLC), mass spectroscopy, or other suitablemeans. U.S. Pat. No. 5,595,900 teaches several methods by which cellswith desired extracellular carbohydrate structures may be identified. Ina preferred embodiment, MALDI-TOF mass spectrometry is used to analyzethe cleaved N-glycans.

Prior to selection of a desired transformant, it may be desirable todeplete the transformed population of cells having undesired phenotypes.For example, when the method is used to engineer a functionalmannosidase activity into cells, the desired transformants will havelower levels of mannose in cellular glycoprotein. Exposing thetransformed population to a lethal radioisotope of mannose in the mediumdepletes the population of transformants having the undesired phenotype,i.e. high levels of incorporated mannose (Huffaker, 1983).Alternatively, a cytotoxic lectin or antibody, directed against anundesirable N-glycan, may be used to deplete a transformed population ofundesired phenotypes (e.g., Stanley, 1977). U.S. Pat. No. 5,595,900teaches several methods by which cells with a desired extracellularcarbohydrate structures may be identified. Repeatedly carrying out thisstrategy allows for the sequential engineering of more and more complexglycans in lower eukaryotes.

To detect host cells having on their surface a high degree of thehuman-like N-glycan intermediate GlcNAcMan₃GlcNAc₂, for example, one mayselect for transformants that allow for the most efficient transfer ofGlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay.This screen may be carried out by growing cells harboring thetransformed library under selective pressure on an agar plate andtransferring individual colonies into a 96-well microtiter plate. Aftergrowing the cells, the cells are centrifuged, the cells resuspended inbuffer, and after addition of UDP-GlcNAc and GnT II, the release of UDPis determined either by HPLC or an enzyme linked assay for UDP.Alternatively, one may use radioactively labeled UDP-GlcNAc and GnT II,wash the cells and then look for the release of radioactive GlcNAc byN-actylglucosaminidase. All this may be carried manually or automatedthrough the use of high throughput screening equipment. Transformantsthat release more UDP, in the first assay, or more radioactively labeledGlcNAc in the second assay, are expected to have a higher degree ofGlcNAcMan₃GlcNAc₂ on their surface and thus constitute the desiredphenotype. Similar assays may be adapted to look at the N-glycans onsecreted proteins as well.

Alternatively, one may use any other suitable screen such as a lectinbinding assay that is able to reveal altered glycosylation patterns onthe surface of transformed cells. In this case the reduced binding oflectins specific to terminal mannoses may be a suitable selection tool.Galantus nivalis lectin binds specifically to terminal α-1,3 mannose,which is expected to be reduced if sufficient mannosidase II activity ispresent in the Golgi. One may also enrich for desired transformants bycarrying out a chromatographic separation step that allows for theremoval of cells containing a high terminal mannose content. Thisseparation step would be carried out with a lectin column thatspecifically binds cells with a high terminal mannose content (e.g.,Galantus nivalis lectin bound to agarose, Sigma, St. Louis, Mo.) overthose that have a low terminal mannose content.

In addition, one may directly create such fusion protein constructs, asadditional information on the localization of active carbohydratemodifying enzymes in different lower eukaryotic hosts becomes availablein the scientific literature. For example, it is known that humanβ1,4-GalTr can be fused to the membrane domain of MNT, amannosyltransferase from S. cerevisiae, and localized to the Golgiapparatus while retaining its catalytic activity (Schwientek et al., J.Biol. Chem. 270:5483-9 1995). If S. cerevisiae or a related organism isthe host to be engineered one may directly incorporate such findingsinto the overall strategy to obtain complex N-glycans from such a host.Several such gene fragments in P. pastoris have been identified that arerelated to glycosyltransferases in S. cerevisiae and thus could be usedfor that purpose.

Alteration of Host Cell Glycosylation Using Fusion Constructs fromCombinatorial Libraries

The construction of a preferred combinatorial DNA library is illustratedschematically in FIG. 2 and described in Example 11. The fusionconstruct may be operably linked to a multitude of vectors, such asexpression vectors well-known in the art. A wide variety of such fusionconstructs were assembled using representative activities as shown inTable 6. Combinations of targeting peptide/catalytic domains may beassembled for use in targeting glycosyltransferase and glycosidase (suchas mannosidase) activities in the ER, Golgi and the trans Golgi networkaccording to the invention. Surprisingly, the same catalytic domain mayhave no effect to a very profound effect on N-glycosylation patterns,depending on the type of targeting peptide used (see, e.g., Table 7,Example 11).

The present invention also provides for the nucleic acid encoding thefusion constructs described herein, vectors comprising such fusionconstructs, and host cells transformed with such fusion constructs.

Mannosidase Fusion Constructs

A representative example of a mannosidase fusion construct derived froma combinatorial DNA library of the invention is pFB8, which a truncatedSaccharomyces SEC12(m) targeting peptide (988-1296 nucleotides of SEC12from SwissProt P11655) ligated in-frame to a 187 N-terminal amino aciddeletion of a mouse α-mannosidase IA (Genbank AN: 6678787). Thenomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces SEC12 (m)/mouse mannosidase IA Δ187. The encoded fusionprotein localizes in the ER by means of the SEC12 targeting peptidesequence while retaining its mannosidase catalytic domain activity andis capable of producing in vivo N-glycans having a Man₅GlcNAc₂ structure(Example 11; FIG. 6F, FIG. 7B).

The fusion construct pGC5, Saccharomyces MNS1(m)/mouse mannosidase IBΔ99, is another example of a fusion construct having intracellularmannosidase trimming activity (Example 11; FIG. 5D, FIG. 8B). Fusionconstruct pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)is yet another example of an efficient fusion construct capable ofproducing N-glycans having a Man₅GlcNAc₂ structure in vivo. By creatinga combinatorial DNA library of these and other such mannosidase fusionconstructs according to the invention, a skilled artisan may distinguishand select those constructs having optimal intracellular trimmingactivity from those having relatively low or no activity. Methods usingcombinatorial DNA libraries of the invention are advantageous becauseonly a select few mannosidase fusion constructs may produce aparticularly desired N-glycan in vivo.

In addition, mannosidase trimming activity may be specific to aparticular protein of interest. Thus, it is to be further understoodthat not all targeting peptide/mannosidase catalytic domain fusionconstructs may function equally well to produce the proper glycosylationon a glycoprotein of interest. Accordingly, a protein of interest may beintroduced into a host cell transfected with a combinatorial DNA libraryto identify one or more fusion constructs which express a mannosidaseactivity optimal for the protein of interest. One skilled in the artwill be able to produce and select optimal fusion construct(s) using thecombinatorial DNA library approach described herein.

It is apparent, moreover, that other such fusion constructs exhibitinglocalized active mannosidase catalytic domains (or more generally,domains of any enzyme) may be made using techniques such as thoseexemplified in Example 11 and described herein. It will be a matter ofroutine experimentation for one skilled in the art to make and use thecombinatorial DNA library of the present invention to optimize, forexample, Man₅GlcNAc₂ production from a library of fusion constructs in aparticular expression vector introduced into a particular host cell.

Glycosyltransferase Fusion Constructs

Similarly, a glycosyltransferase combinatorial DNA library was madeusing the methods of the invention. A combinatorial DNA library ofsequences derived from glycosyltransferase I (GnTI) activities wereassembled with targeting peptides and screened for efficient productionin a lower eukaryotic host cell of a GlcNAcMan₅GlcNAc₂ N-glycanstructure on a marker glycoprotein. A fusion construct shown to produceGlcNAcMan₅GlcNAc₂ (pPB104), Saccharomyces MNN9(s)/human GnTI Δ38 wasidentified (Example 15). A wide variety of such GnTI fusion constructswere assembled (Example 15, Table 10). Other combinations of targetingpeptide/GnTI catalytic domains can readily be assembled by making acombinatorial DNA library. It is also apparent to one skilled in the artthat other such fusion constructs exhibiting glycosyltransferaseactivity may be made as demonstrated in Example 15. It will be a matterof routine experimentation for one skilled in the art to use thecombinatorial DNA library method described herein to optimizeGlcNAcMan₅GlcNAc₂ production using a selected fusion construct in aparticular expression vector and host cell line.

As stated above for mannosidase fusion constructs, not all targetingpeptide/GnTI catalytic domain fusion constructs will function equallywell to produce the proper glycosylation on a glycoprotein of interestas described herein. However, one skilled in the art will be able toproduce and select optimal fusion construct(s) using a DNA libraryapproach as described herein. Example 15 illustrates a preferredembodiment of a combinatorial DNA library comprising targeting peptidesand GnTI catalytic domain fusion constructs involved in producingglycoproteins with predominantly GlcNAcMan₅GlcNAc₂ structure. Example 17discloses the use of a sialyltransferase fusion construct and twodifferent GnTII and MannII fusion constructs.

Using Multiple Fusion Constructs to Alter Host Cell Glycosylation

In another example of using the methods and libraries of the inventionto alter host cell glycosylation, a P. pastoris strain with an OCH1deletion that expresses a reporter protein (K3) was transformed withmultiple fusion constructs isolated from combinatorial libraries of theinvention to convert high mannose N-glycans to human-like N-glycans(Example 15). First, the mannosidase fusion construct pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed intoa P. pastoris strain lacking 1,6 initiating mannosyltransferasesactivity (i.e. och1 deletion; Example 1). Second, pPB103 comprising a K.lactis MNN2-2 gene (Genbank AN: AF106080) encoding an UDP-GlcNActransporter was constructed to increase further production ofGlcNAcMan₅GlcNAc₂. The addition of the UDP-GlcNAc transporter increasedproduction of GlcNAcMan₅GlcNAc₂ significantly in the P. pastoris strainas illustrated in FIG. 10B. Third, pPB104 comprising Saccharomyces MNN9(s)/human GnTI Δ38 was introduced into the strain. This P. pastorisstrain is referred to as “PBP-3.”

It is understood by one skilled in the art that host cells such as theabove-described yeast strains can be sequentially transformed and/orco-transformed with one or more expression vectors. It is alsounderstood that the order of transformation is not particularly relevantin producing the glycoprotein of interest. The skilled artisanrecognizes the routine modifications of the procedures disclosed hereinmay provide improved results in the production of the glycoprotein ofinterest.

The importance of using a particular targeting peptide sequence with aparticular catalytic domain sequence becomes readily apparent from theexperiments described herein. The combinatorial DNA library provides atool for constructing enzyme fusions that are involved in modifyingN-glycans on a glycoprotein of interest, which is especially useful inproducing human-like glycoproteins. (Any enzyme fusion, however, may beselected using libraries and methods of the invention.) Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce K3 with N-glycans of the structure Man₅GlcNAc₂as shown in FIGS. 5D and 5E. This confers a reduced molecular mass tothe cleaved glycan compared to the K3 of the parent OCH1 deletionstrain, as was detected by MALDI-TOF mass spectrometry in FIG. 5C.

Similarly, the same approach was used to produce another secretedglycoprotein: IFN-β comprising predominantly Man₅GlcNAc₂. TheMan₅GlcNAc₂ was removed by PNGase digestion (Papac et al. 1998) andsubjected to MALDI-TOF as shown in FIG. 6A-6F. A single prominent peakat 1254 (m/z) confirms Man₅GlcNA₂ production on IFN-β in FIG. 6E (pGC5)(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99) and 6F (pFB8)(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187). Furthermore, in theP. pastoris strain PBP-3 comprising pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187), pPB104 (Saccharomyces MNN9 (s)/human GnTI Δ38) andpPB103 (K. lactis MNN2-2 gene), the hybrid N-glycan GlcNAcMan₅GlcNAc₂[b] was detected by MALDI-TOF (FIG. 10).

After identifying transformants with a high degree of mannose trimming,additional experiments were performed to confirm that mannosidase(trimming) activity occurred in vivo and was not predominantly theresult of extracellular activity in the growth medium (Example 13; FIGS.7-9).

Sequential Glycosylation Reactions

In a preferred embodiment, such targeting peptide/catalytic domainlibraries are designed to incorporate existing information on thesequential nature of glycosylation reactions in higher eukaryotes.Reactions known to occur early in the course of glycoprotein processingrequire the targeting of enzymes that catalyze such reactions to anearly part of the Golgi or the ER. For example, the trimming ofMan₈GlcNAc₂ to Man₅GlcNAc₂ by mannosidases is an early step in complexN-glycan formation. Because protein processing is initiated in the ERand then proceeds through the early, medial and late Golgi, it isdesirable to have this reaction occur in the ER or early Golgi. Whendesigning a library for mannosidase I localization, for example, onethus attempts to match ER and early Golgi targeting signals with thecatalytic domain of mannosidase I. As another example, the sialylationof glycoproteins occurs in Golgi. Thus, when designing a library forexpression of sialyltransferase, one thus attempts to match Golgitargeting signals with the catalytic domain of a sialyltransferase.

Codon Optimization and Nucleotide Substitution

The methods of the invention may be performed in conjunction withoptimization of the base composition for efficienttranscription/translation of the encoded protein in a particular host,such as a fungal host. This includes codon optimization to ensure thatthe cellular pools of tRNA are sufficient. The foreign genes (ORFs) maycontain motifs detrimental to complete transcription/translation in thefungal host and, thus, may require substitution to more amenablesequences. The expression of each introduced protein can be followedboth at the transcriptional and translational stages by well knownNorthern and Western blotting techniques, respectively (Sambrook, J. andRussell, D. W., 2001).

Vectors

In another aspect, the present invention provides vectors (includingexpression vectors), comprising genes encoding activities glycosylationenzymes, a promoter, a terminator, a selectable marker and targetingflanking regions. Such promoters, terminators, selectable markers andflanking regions are readily available in the art. In a preferredembodiment, the promoter in each case is selected to provide optimalexpression of the protein encoded by that particular ORF to allowsufficient catalysis of the desired enzymatic reaction. This steprequires choosing a promoter that is either constitutive or inducible,and provides regulated levels of transcription. In another embodiment,the terminator selected enables sufficient termination of transcription.In yet another embodiment, the selectable markers used are unique toeach ORF to enable the subsequent selection of a fungal strain thatcontains a specific combination of the ORFs to be introduced. In afurther embodiment, the locus to which each fusion construct (encodingpromoter, ORF and terminator) is localized, is determined by the choiceof flanking region. The present invention is not limited to the use ofthe vectors disclosed herein.

Integration Sites

As one ultimate goal of this genetic engineering effort is a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the host(e.g., fungal) chromosome preferably involves careful planning Theengineered strain may likely have to be transformed with a range ofdifferent genes, and these genes will have to be transformed in a stablefashion to ensure that the desired activity is maintained throughout thefermentation process. As described herein, any combination of variousdesired enzyme activities may be engineered into the fungal proteinexpression host, e.g., sialyltransferases, mannosidases,fucosyltransferases, galactosyltransferases, glucosyltransferases,GlcNAc transferases, ER and Golgi specific transporters (e.g. syn andantiport transporters for UDP-galactose and other precursors), otherenzymes involved in the processing of oligosaccharides, and enzymesinvolved in the synthesis of activated oligosaccharide precursors suchas UDP-galactose, CMP-N-acetylneuraminic acid. Genes which encodeenzymes known to be characteristic of non-human glycosylation reactionsin fugal hosts and their corresponding proteins have been extensivelycharacterized in a number of lower eukaryotes (e.g., Saccharomycescerevisiae, Trichoderma reesei, Aspergillus nidulans, P. pastoris,etc.), thereby providing a list of known glycosyltransferases in lowereukaryotes, their activities and their respective genetic sequence.These genes are likely to be selected from the group ofmannosyltransferases e.g., 1,3 mannosyltransferases (e.g., MNN1 in S.cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g., the KTR/KREfamily from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S.cerevisiae), mannosylphosphate transferases and their regulators (MNN4and MNN6 from S. cerevisiae) and additional enzymes that are involved inaberrant (i.e. non-human) glycosylation reactions. Examples of preferredmethods for modifying glycosylation in a lower eukaryotic host cell,such as Pichia pastoris, are shown in Table 6. (The HDEL and KDEL signalpeptides in the second row of the third column are shown in SEQ ID NOS:5 and 6, respectively).

TABLE 6 Some Preferred Embodiments For Modifying Glycosylation In ALower Eukaroytic Microorganism Suitable Suitable Suitable Sources ofSuitable Transporters Desired Catalytic Localization Gene and/orStructure Activities Sequences Deletions Phosphatases Man₅GlcNAc₂ α-1,2-Mns1 OCH1 none mannosidase (N-terminus, MNN4 (murine, S.cerevisiae) MNN6human, Och1 Bacillus sp., (N-terminus, A.nidulans ) S.cerevisiae,P.pastoris) Ktr1 Mnn9 Mnt1 (S.cerevisiae) KDEL, HDEL (C-terminus)GlcNAcMan₅ GlcNAc Och1 OCH1 UDP- GlcNAc₂ Transferase (N-terminus, GlcNAcI, (human, S.cerevisiae, MNN4 transporter murine, P.pastoris) MNN6(human, rat etc.) KTR1 murine, (N-terminus) K.lactis) Mnn1 UDPase(N-terminus, (human) S.cerevisiae) Mnt1 (N-terminus, S.cerevisiae)GDPase (N-terminus, S.cerevisiae) GlcNAcMan₃ mannosidase Ktr1 OCH1 UDP-GlcNAc₂ II Mnn1 MNN4 GlcNAc (N-terminus, MNN6 transporter S.cerevisiae)(human, Mnt1 murine, (N-terminus, K.lactis) S. cerevisiae) UDPaseKre2/Mnt1 (human) (S.cerevisiae) Kre2 (P.pastoris) Ktr1 (S.cerevisiae)Ktr1 (P.pastoris) Mnn1 (S.cerevisiae) GlcNAc₍₂₋₄₎ GlcNAc Mnn 1 OCH1 UDP-Man₃GlcNAc₂ Transferase (N-terminus, MNN4 GlcNAc II, III, IV, VS.cerevisiae) MNN6 transporter (human, Mnt1 (human, murine) (N-terminus,murine, S.cerevisiae) K.lactis) Kre2/Mnt1 UDPase (S.cerevisiae) (human)Kre2 (P.pastoris) Ktr1 (S.cerevisiae) Ktr1 (P.pastoris) Mnn1(S.cerevisiae) Gal₍₁₋₄₎ β-1,4- Mnn1 OCH1 UDP- GlcNAc₍₂₋₄₎- Galactosyl(N-terminus, MNN4 Galactose Man₃GlcNAc₂ transferase S.cerevisiae) MNN6transporter (human) Mnt1 (human, (N-terminus, S.pombe) S.cerevisiae)Kre2/Mnt1 (S.cerevisiae) Kre2 (P.pastoris) Ktr1 (S.cerevisiae) Ktr1(P.pastoris) Mnn1 (S.cerevisiae) NANA₍₁₋₄₎- α-2,6- KTR1 OCH1 CMP-SialicGal₍₁₋₄₎ Sialyl- MNN1 MNN4 acid GlcNAc₍₂₋₄₎- transferase (N-terminus,MNN6 transporter Man₃GlcNAc₂ (human) S.cerevisiae) (human) α-2,3- MNT1Sialyl- (N-terminus, transferase S.cerevisiae) Kre2/Mnt1 (S.cerevisiae)Kre2 (P.pastoris) Ktr1 (S.cerevisiae) Ktr1 (P.pastoris) MNN1(S.cerevisiae) MNN2 (S.cerevisiae)

Methods for Producing CMP-Sia for the Generation of RecombinantN-Glycans

The present invention provides methods for production of a functionalCMP-Sia biosynthetic pathway in a host cell that lacks endogenousCMP-Sia, such as a fungal cell. The present invention also provides amethod for creating a host cell that has been modified to express a newor altered CMP-Sia pathway. The invention further provides a method forcreating a host cell that comprises a cellular pool of CMP-Sia.

The methods involve the cloning and expression in a host cell of severalgenes encoding enzymes of the CMP-Sia biosynthetic pathway resulting ina cellular pool of CMP-Sia in the host cell which can be utilized in theproduction of sialylated glycans on proteins of interest. In general,the addition of sialic acids to glycans requires the presence of thesialyltransferase, a glycan acceptor (e.g., Gal₂GlcNAc₂Man₃GlcNAc₂) andthe sialyl donor molecule, CMP-Sia. The synthesis of the CMP-Sia donormolecule in higher organisms (e.g., mammals) is a four enzyme, multiplereaction process starting with the substrate UDP-GlcNAc and resulting inCMP-Sia (FIG. 14A). The process initiates in the cytoplasm producingsialic acid which is then translocated into the nucleus where Sia isconverted to CMP-Sia by CMP-sialic acid synthase. Subsequently, CMP-Siaexits the nucleus into the cytoplasm and is then transported into theGolgi where sialyltransferases catalyze the transfer of sialic acid ontothe acceptor glycan. In contrast, the bacterial pathway for synthesizingCMP-Sia from UDP-GlcNAc involves only three enzymes and twointermediates (FIG. 14), with all reactions occurring in the cytoplasm.

Accordingly, the methods of the invention involve generating a pool ofCMP-Sia in a non-human host cell that lacks endogenous CMP-Sia byintroducing a functional CMP-Sia biosynthetic pathway. With readilyavailable DNA sequence information from genetic databases (e.g.,GenBank, Swissprot), enzymes and/or activities involved in the CMP-Siapathways (Example 16) are cloned. Using standard techniques known tothose skilled in the art, nucleic acid molecules encoding one or moreenzymes (or catalytically active fragments thereof) involved in thebiosynthesis or transport of CMP-Sia are inserted into appropriateexpression vectors under the transcriptional control of promoters and/orother expression control sequences capable of driving transcription in aselected host cell of the invention (e.g., a fungal host cell). Thefunctional expression of such enzymes in the selected host cells of theinvention can be detected. In one embodiment, the functional expressionof such enzymes in the selected host cells of the invention can bedetected by measuring the intermediate formed by the enzyme. The methodsof the invention are not limited to the use of the specific enzymesources disclosed herein. In certain preferred embodiments,combinatorial libraries of the invention are utilized to select and/oroptimize CMP-Sia pathway activity in the selected host cell.

Engineering a Mammalian CMP-Sialic Acid Biosynthetic Pathway in a HostCell

In one embodiment of the invention, the method involves cloning severalgenes encoding enzymes in the CMP-Sia biosynthetic pathway, includingUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase,N-acetylneuraminate-9-phosphate synthase,N-acetylneuraminate-9-phosphatase, CMP-sialic acid phosphatase andCMP-sialic acid synthase, and expressing one or more of said clonedgenes in a host cell which lacks endogenous CMP-Sia, such as a fungalhost cell. The genes are expressed to generate each enzyme, producingintermediates that are used for subsequent enzymatic reactions. Example16 describe methods for the introduction of these enzymes into a fungalhost (e.g., P. pastoris) using a selection marker. Alternatively, theenzymes are expressed together to produce or increase downstreamintermediates whereby subsequent enzymes are able to act upon them.

The first enzyme in the pathway is a bi-functional enzyme that is both aUDP-GlcNAc epimerase and an N-acetylmannosamine kinase, convertingUDP-GlcNAc through N-acetylmannosamine (ManNAc) toN-acetylmannosamine-6-phosphate (ManNAc-6-P) (Hinderlich, 1997). Thisenzyme was originally cloned from a rat liver cDNA library (Stasche,1997). Homologs to this enzyme have subsequently been found to exist inother species, for example, human (NM_(—)005476); E. coli (Ringenberg etal. 2003); Mannheimia haemolytica (McKerrell and Lo, Infect Immun. 2002May; 70(5):2622-9). In a preferred embodiment, a gene encoding thefunctional UDP-N-acetylglucosamine-2-epimerase enzyme, includinghomologs, variants and derivatives thereof, is cloned and expressed in ahost cell (for example, a non-human host cell which lacks endogenousCMP-Sia, such as a fungal host cell). In another preferred embodiment, agene encoding the functional N-acetylmannosamine kinase enzyme,including homologs, variants and derivatives thereof, is cloned andexpressed in a host cell, such as a fungal host cell. In a morepreferred embodiment, a gene encoding the bifunctionalUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase enzyme,including homologs, variants and derivatives thereof, is cloned andexpressed in a host cell, such as a fungal host cell (e.g., P.pastoris). The functional expression of these genes can be detectedusing a functional assay. In one embodiment, the functional expressionof such genes can be detected by monitoring the formation of ManNAc andManNAc-6-P intermediates.

The second enzyme in the pathway, N-acetylneuraminic acid phosphatesynthase, was cloned from human liver based on its homology to the E.coli sialic acid synthase gene, NeuB (Lawrence, 2000). Homologs of theN-acetylneuraminic acid phosphate synthase enzyme are also known in ratliver (Chen et al., Glycobiology. 2002 12(2):65-71) and mouse (Nakata etal., Biochem. Biophys. Res. Commun. 2000; 273(2):642-8). This enzymecatalyzes the conversion of ManNAc-6-P to sialate 9-phosphate (alsoreferred to as Sia-9P, N-acetylneuraminate 9-phosphate, or Neu5Ac-9P).Accordingly, in a preferred embodiment, a gene encoding the functionalN-acetylneuraminate 9-phosphate synthase enzyme, including homologs,variants and derivatives thereof, is cloned and expressed in a host cell(for example a non-human host cell which lacks endogenous CMP-Sia, suchas a fungal host cell). The expression of N-acetylneuraminic acidphosphate synthase in the host can be detected using a functional assay.In one embodiment, the functional expression of N-acetylneuraminic acidphosphate synthase can be detected by monitoring the formation ofSia-9P.

The third enzyme in the pathway, N-acetylneuraminate 9-phosphatase(Sia-9-phosphatase), is involved in the conversion of Sia-9-P to sialicacid. The cloning of this enzyme has recently been reported by Maliekalet al., 2006 (See GenBank AN NM_(—)152667). Although the activity ofthis enzyme has been detected in mammalian cells, no such activity hasbeen identified in fungal cells. Therefore, the lack ofSia-9-phosphatase would cause a break in the pathway. Accordingly, in apreferred embodiment, the method of the present invention involvesisolating and cloning a Sia-9-phosphatase gene and expressing it in anon-human host cell, such as a fungal host cell. (See Example 16.) Suchhosts include, e.g., yeast, fungal, insect and bacterial cells. In amore preferred embodiment, the Sia-9-phosphatase gene, includinghomologs, variants and derivatives thereof, is expressed in a non-humanhost cell that lacks endogenous CMP-Sia or that expresses inadequatelevels of CMP-Sia, such as a fungal host. The expression ofSia-9-phosphatase in the host can be detected using a functional assay.In one embodiment, the functional expression of Sia-9-phosphatase can bedetected by monitoring the formation of sialic acid.

The next enzyme in the mammalian pathway, CMP-Sia synthase, wasoriginally cloned from the murine pituitary gland by functionalcomplementation of a cell line deficient in this enzyme (Munster, 1998).Homologs have been found to exist in E. coli (Mercker and Troy, 1990);and human (Raju et al., 2001). This enzyme converts sialic acid toCMP-Sia, which is the donor substrate in a sialyltransferase reaction inthe Golgi. Accordingly, in an even more preferred embodiment, a geneencoding the functional CMP-Sia synthase enzyme, including homologs,variants and derivatives thereof, is cloned and expressed in a host cell(for example a non-human that lacks endogenous CMP-Sia, such as a fungalhost cell). The expression of CMP-Sia synthase in the host can bedetected using a functional assay. In one embodiment, the functionalexpression of CMP-Sia synthase can be detected by monitoring theformation of CMP-Sia.

The method of the present invention further involves the production ofthe intermediates produced in a host cell as a result of expressing theabove enzymes in the CMP-Sia pathway. Preferably, the intermediatesproduced include one or more of the following: UDP-GlcNAc, ManNAc,ManNAc-6-P, Sia-9-P, Sia and CMP-Sia. Additionally, each intermediateproduced by the enzymes is preferably detected. For example, to detectthe presence or absence of an intermediate, an assay as described inExample 16 is used. Accordingly, the invention also provides assays todetect the N-glycan intermediates produced in a non-human host cell thatlacks endogenous CMP-Sia, such as a fungal host cell.

A skilled artisan recognizes that the mere availability of one or moreenzymes in the CMP-sialic acid biosynthetic pathway does not suggestthat such enzymes can be functionally expressed in a host cell thatlacks endogenous CMP-Sia, such as a fungal host cell. To date, theability of such host cell to express these mammalian enzymes to create afunctional de novo CMP-Sia biosynthetic pathway has not been described.The present invention provides for the first time the functionalexpression of at least one mammalian enzyme involved in CMP-Siabiosynthesis in a fungal host: the mouse CMP-Sia synthase (Example 16),indicating that production of CMP-Sia via the mammalian pathway (inwhole or in part) is possible in a fungal host and thus in othernon-human hosts that lack endogenous CMP-Sia.

The invention described herein is not limited to the use of the specificenzymes, genes, plasmids and constructs disclosed herein. A person ofskill would readily understand how to use any homologs, variants,derivatives and functional equivalents of the genes involved in thesynthesis of CMP-Sia.

To produce sialylated, recombinant glycoproteins in a host cell thatlacks endogenous CMP-Sia (e.g., a fungal host such as P. pastoris) or ina host cell that needs or can benefit from increased levels of CMP-Sia,the above mentioned mammalian enzymes can be expressed using acombinatorial DNA library approach as disclosed herein, generating apool of CMP-Sia, which is transferred onto galactosylated N-glycans inthe presence of a sialyltransferase. Accordingly, the present inventionprovides a method for engineering a CMP-Sia biosynthetic pathway into ahost cell by expressing one or more of each of the enzymes such thatthey function, preferably so that they function optimally, in thesubcellular location to which they are targeted in the selected hostcell. Mammalian, bacterial or hybrid engineered CMP-Sia biosyntheticpathways are provided.

Engineering a Bacterial CMP-Sialic Acid Biosynthetic Pathway in a HostCell

The metabolic intermediate UDP-GlcNAc is common to eukaryotes andprokaryotes, providing an endogenous substrate from which to initiatethe synthesis of CMP-Sia (FIG. 14). Based on the presence of this commonintermediate, the CMP-Sia biosynthetic pathway can be engineered intonon-human host cells that lack endogenous CMP-Sia by introducing thegenes encoding the bacterial UDP-GlcNAc epimerase (NeuC), sialatesynthase (NeuB) and CMP-Sia (NeuA) synthase into the host cell, e.g., bytransformation methods such as on plasmids, and preferably, byintegration into the host cell chromosome. Accordingly, another aspectof the present invention involves engineering a bacterial CMP-Siabiosynthetic pathway into host cells that lack an endogenous CMP-Siapathway or into host cells that need or would benefit from increasedlevels of CMP-Sia. The expression of bacterial Neu genes in cells thatlack an endogenous CMP-Sia biosynthetic pathway enables the generationof a cellular CMP-Sia pool, which can subsequently facilitate theproduction of recombinant N-glycans having detectable level ofsialylation on a protein of interest, such as recombinantly expressedglycoproteins. The bacterial enzymes involved in the synthesis ofCMP-Sia include UDP-GlcNAc epimerase (NeuC), sialate synthase (NeuB) andCMP-Sia synthase (NeuA). In one embodiment, the NeuC, NeuB, and NeuAgenes, which encode these functional enzymes, respectively, includinghomologs, variants and derivatives thereof, are cloned and expressed innon-human host cells that lack an endogenous CMP-Sia pathway, such as afungal host cell. The sequences of NeuC, NeuB and NeuA genes are shownin FIGS. 15-17, respectively. The expression of these genes in the hostcell generates the intermediate molecules in the biosynthetic pathway ofCMP-sialic acid (FIG. 14B).

In addition to these three enzymes, the method for synthesizing thebacterial CMP-Sia biosynthetic pathway from UDP-GlcNAc involvesgenerating two intermediates: ManNAc and Sia (FIG. 14B). The conversionof UDP-GlcNAc to ManNAc is facilitated by the NeuC gene. The conversionof ManNAc to Sia is facilitated by the NeuB gene and the conversion ofSia to CMP-Sia is facilitated by the NeuA gene. These three enzymes (orhomologs thereof) have thus far been found linked together in pathogenicbacteria—i.e., not single gene has been found without the other two.Introducing the bacterial pathway into a host cell, such as a fungalhost, thus requires the manipulation of fewer genes than introducingmammalian pathway.

The E. coli UDP-GlcNAc epimerase, encoded by the E. coli NeuC gene, isthe first enzyme involved in the bacterial synthesis of polysialic acid(Ringenberg, 2001). The NeuC gene (Genbank AN: M84026.1; SEQ ID NO:57)encoding this enzyme was isolated from the pathogenic E. coli K1 strainand encodes a protein of 391 amino acids (SEQ ID NO:58) (FIG. 15)(Zapata, 1992). The encoded UDP-GlcNAc epimerase catalyzes theconversion of UDP-GlcNAc to ManNAc. Homologs of this enzyme have beenidentified in several pathogenic bacteria, including Streptococcusagalactiae, Synechococcus sp. WH 8102, Clostridium thermocellum, Vibriovulnificus, Legionella pnuemophila, and Campylobacter jejuni. In oneembodiment, a gene encoding the functional E. coli UDP-GlcNAc epimeraseenzyme (NeuC), including homologs, variants and derivatives thereof, iscloned and expressed in a host cell (for example a non-human host cell,such as a fungal host). The expression of NeuC in the host can bedetected using a functional assay. In one embodiment, the functionalexpression NeuC can be detected by monitoring the formation of ManNAc.

The second enzyme in the bacterial pathway is sialate synthase, whichdirectly converts ManNAc to Sia, bypassing several enzymes andintermediates present in the mammalian pathway. This enzyme of 346 aminoacids (SEQ ID NO:60), is encoded by the E. coli NeuB gene (Genbank AN:U05248.1; SEQ ID NO:59) (FIG. 16) (Annunziato, 1995). In anotherembodiment, a gene encoding a functional E. coli sialate synthase enzyme(NeuB), including homologs, variants and derivatives thereof, is clonedand expressed in a host cell (for example, a non-human host cell, suchas a fungal host cell). The expression of NeuB in the host can bedetected using a functional assay. In one embodiment, the functionalexpression NeuB can be detected by monitoring the formation of Sia.

The third enzyme in this bacterial pathway is CMP-Sia synthase,consisting of 419 amino acids (SEQ ID NO:62) and encoded by the E. coliNeuA gene (Genbank AN: J05023; SEQ ID NO:61) (FIG. 17). CMP-Sia synthaseconverts Sia to CMP-Sia (Zapata, 1989). The NeuA gene is found in thesame organisms as the NeuC and NeuB genes. Accordingly, in yet anotherembodiment, a gene (NeuA) encoding a functional E. coli CMP-Sia synthaseenzyme, including homologs, variants and derivatives thereof, is clonedand expressed in a host cell (for example, a non-human host cell, suchas a fungal host cell). In one embodiment, the expression NeuA can bedetected by monitoring the formation of CMP-Sia.

In yet another embodiment, the gene encoding a functional bacterialCMP-Sia synthase (e.g. NeuA) encodes a fusion protein comprising a:catalytic domain having the activity of a bacterial CMP-Sia synthase anda cellular targeting signal peptide (not normally associated with thecatalytic domain) selected to target the enzyme to the nucleus of thehost cell. In one embodiment, said cellular targeting signal peptidecomprises a domain of the SV40 capside polypeptide VP1. In anotherembodiment, the signal peptide comprises one or more endogenoussignaling motifs from a mammalian CMP-Sia synthase that ensure correctlocalization of the enzyme to the nucleus. Methods for making saidfusion protein are well known in the art.

After PCR amplification of the E. coli NeuA, NeuB and NeuC genes, theamplified fragments were ligated into a selectable yeast integrationvector under the control of a promoter (Example 16). After transforminga host strain (e.g., P. pastoris), with each vector carrying the Neugene fragments, colonies were screened by applying positive selectionfor the presence of vectors and then screening transformants for Neugene enzymatic activity. The ability of a non-human host cell that lacksendogenous sialylation to express the bacterial enzymes involved increating a de novo CMP-Sia biosynthetic pathway is provided for thefirst time by this invention.

Engineering a Hybrid Mammalian/Bacterial CMP-Sialic Acid BiosyntheticPathway in a Host Cell

Both mammalian and bacterial CMP-Sia biosynthetic pathways require thatboth CTP and sialic acid be available to the CMP-Sia synthase. Althoughsimilar in enzymatic function to the corresponding bacterial enzyme, themammalian CMP-Sia synthase may include one or more endogenous signalingmotifs that ensure correct localization to the nucleus. Becauseeukaryotes have a nucleus-localized pool of CTP and the prokaryoticCMP-Sia synthase may not localize to this compartment, a hybrid CMP-Siabiosynthetic pathway combining both mammalian and bacterial enzymes is apreferred method for the production of sialic acid and its intermediatesin a non-human host cell that lacks endegenous sialylation, such as afungal host cell. To this end, a pathway can be engineered into the hostcell which involves the integration of both NeuC and NeuB as well as amammalian CMP-Sia synthase. The CMP-Sia synthase enzyme may be selectedfrom several mammalian homologs that have been cloned and characterized(Genbank AN: AJ006215; SEQ ID NO:63) (Munster, 1998) (see e.g., themurine CMP-Sia synthase) (FIG. 18). In one preferred embodiment, thehost cell is transformed with UDP-GlcNAc epimerase (E. coli NeuC) andsialate synthase (E. coli NeuB) in combination with the mouse CMP-Siasynthase. The host engineered with this hybrid CMP-Sia biosyntheticpathway produces a cellular pool of the donor molecule CMP-Sia (FIG.25). In a more preferred embodiment, the combination of the enzymesexpressed in the host is selected for enhanced production of the donormolecule CMP-Sia.

Engineering Enzymes Involved in Alternative Routes for Enhancing theProduction of CMP-Sialic Acid Pathway Intermediates in Host Cells

In yet another aspect of the invention, enzymes involved in alternatepathways of CMP-sialic acid biosynthesis are engineered into non-humanhost cells that lack endogenous sialylation, such as fungal host cells;or in host cells in which endogenous sialylation may be altered, e.g.,enhanced. For example, it is contemplated that when an intermediatebecomes limiting during one of the methods outlined above, theintroduction of an enzyme that uses an alternate mechanism to producethat intermediate will serve as a sufficient substitute in theproduction of CMP-sialic acid, or any intermediate along this pathway.Embodiments are described herein for the production of the intermediatesManNAc and Sia, though this approach may be extended to produce otherintermediates. Furthermore, any of these enzymes can be incorporatedinto either the mammalian, bacterial or hybrid pathways, either in theabsence of the enzymes mentioned previously (i.e., enzymes producing thesame intermediate) or in the presence of enzymes mentioned previously,i.e., to enhance overall production.

In the above mentioned embodiments, ManNAc is produced from UDP-GlcNAcby either the mammalian enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase orby the bacterial enzyme NeuC. The substrate for this reaction,UDP-GlcNAc, is predicted to be present in sufficient quantities in cellsfor the synthesis of CMP-Sia due to its requirement in producing severalclasses of molecules, including endogenous N-glycans. However, if ManNAcdoes become limiting—potentially due to the increased demand for ManNAcfrom the sialic acid biosynthetic pathway—then the cellular supply ofManNAc may be increased by introducing a GlcNAc epimerase which reactswith the substrate GlcNAc to produce ManNAc.

Accordingly, in one embodiment, a gene encoding a functional GlcNAcepimerase enzyme, including homologs, variants and derivatives thereof,is cloned and expressed in a host cell. Using GlcNAc epimerase todirectly convert GlcNAc to ManNAc is a shorter, more efficient approachcompared with the two-step process involving the synthesis of UDP-GlcNAc(FIG. 19). The GlcNAc epimerase is readily available and, to date, theonly confirmed GlcNAc epimerase to have been cloned is from the pigkidney (Maru, 1996) (Example 16). The gene (Genbank AN: D83766; SEQ IDNO: 65) isolated from pig kidney encodes a protein of 402 amino acids(SEQ ID NO: 64) (FIG. 20). When this enzyme was cloned, it was found tobe identical to the pig renin-binding protein cloned previously (Inoue,1990). Although this is the only protein with confirmed GlcNAc epimeraseactivity, several other renin-binding proteins have been isolated fromother organisms, including humans, mouse, rat and bacteria, amongothers. All are shown to have significant homology. For example, thehuman GlcNAc epimerase homolog (Genbank AN: D10232.1) has 87% identityand 92% similarity to the pig GlcNAc epimerase protein. Although thesehomologs are very similar in sequence, the pig protein is the only onehaving demonstrable epimerase activity to date. The methods of theinvention may be performed using any gene encoding a functional GlcNAcepimerase activity. Based on the presence of GlcNAc epimerase activity,the cloning and expression of this gene in a non-human host cell, suchas a fungal host cell, is predicted to enhance the cellular levels ofManNAc, thereby, providing sufficient substrate for the enzymes thatutilize ManNAc in the CMP-sialic acid biosynthetic pathway.

In another embodiment, sialate aldolase is used to increase cellularlevels of sialic acid, as illustrated in FIG. 21. This enzyme (alsoknown as sialate lyase and sialate pyruvate-lyase) directly catalyzesthe reversible reaction of ManNAc to sialic acid. In the presence of lowconcentrations of Sia, this enzyme catalyzes the condensation of ManNAcand pyruvate to produce Sia. Conversely, when Sia concentrations arehigh, the enzyme causes the reverse reaction to proceed, producingManNAc and pyruvate (Vimr, 1985). In the above embodiments, the presenceof CMP-Sia synthase converts substantially all Sia to CMP-Sia, thusshifting the equilibrium of the aldolase reaction to the condensation ofManNAc and pyruvate to produce Sia. Preferably, the sialate aldolaseused in this embodiment is expressed from the E. coli NanA gene, but theinvention is not limited to this enzyme source. The gene (Genbank AN:X03345; SEQ ID NO:67) for this enzyme encodes a 297 amino acid protein(SEQ ID NO:68) (FIG. 22) (Ohta, 1985). Close homologs to this enzyme arefound in many pathogenic bacteria, including, Salmonella typhimurium,Staphylococcus aureus, Clostridium perfringens, Haemophilus influenzaeamong others. In addition, homologs are also present in mammals,including mice and humans. In one embodiment, cloning a gene encoding asialate aldolase activity and expressing it in a fungal host cellenhances the cellular levels of Sia, thereby providing sufficientsubstrate for the enzymes that utilize Sia in the CMP-sialic acidbiosynthetic pathway (Example 16).

Regulation of CMP-Sialic Acid Synthesis: Feedback Inhibition andInducible Promoters

In mammalian cells, the production of CMP-sialic acid is highlyregulated. CMP-sialic acid acts as a feedback inhibitor, acting onUDP-GlcNAc epimerase/ManNAc kinase to prevent further production ofCMP-Sia (Hinderlich, 1997; Keppler, 1999). In contrast, the bacterialCMP-Sia biosynthetic pathway (FIG. 14B) does not appear to have afeedback inhibitory control mechanism that would limit the production ofCMP-Sia (Ringenberg, 2001). However, incorporation of the E. colisialate aldolase into one of the pathways mentioned above could cause ashift in the direction of the reaction that it catalyzes, depending onthe balance of the equilibrium, thus potentially causing hydrolysis ofSia back to ManNAc. Accordingly, the methods involving sialate aldolaseas outlined above will prevent this reverse reaction from occurring,given the presence of CMP-sialate synthase, which rapidly converts Siato CMP-Sia.

The embodiments described thus far have detailed the constitutiveoverexpression of the enzymes in a particular biosynthetic pathway ofCMP-Sia. Though no literature is currently available that suggests thatthe presence of any of the mentioned intermediates, and/or the finalproduct could be detrimental to a non-human host, such as a fungal host,a preferred embodiment of the invention has one or more of the enzymesunder the control of a regulatable (e.g., an inducible) promoter. Inthis embodiment, the gene (or ORF) encoding the protein of interest(including but not limited to: UDP-GlcNAc 2-epimerase/ManNAc kinase,NeuC, and GlcNAc epimerase) is cloned downstream of an inducible orregulatable promoter (including but not limited to: the alcohol oxidasepromoter (AOX1 or AOX2; Tschopp, 1987), galactose-inducible promoter(GAL10; Yocum, 1984), tetracycline-inducible promoter (TET; Belli,1998)) to facilitate the controlled expression of that enzyme, and thusto regulate the production of CMP-Sia.

Detection of CMP-Sialic Acid and the Intermediate Compounds in itsSynthesis

The methods of the present invention provide engineered pathways toproduce a cellular pool of CMP-Sia in non-human host cells that lack anendogenous CMP-Sia biosynthetic pathway, and/or to enhance production ofCMP-Sia in host cells that have an endegenous pathway. To assess theproduction of each intermediate in the pathway, these intermediates mustbe detectable. Accordingly, the present invention also provides a methodfor detecting such intermediates. A method for detecting a cellular poolof CMP-Sia, for example, is provided in Example 16). Currently, theliterature describes only a few methods for measuring cellular CMP-Siaand its precursors. Early methods involved paper chromatography andthiobarbituric acid analysis and were found to be complicated and timeconsuming (Briles, 1977; Harms, 1973). HPLC (high pressure liquidchromatography) has also been used, though earlier methods employed acidelution resulting in the rapid hydrolysis of the CMP-Sia (Rump, 1986).Most recently, a more robust method has been described usinghigh-performance anion-exchange chromatography using an alkaline elutionprotocol combined with pulsed amperometric detection (HPAEC-PAD)(Fritsch, 1996). This method, in addition to detecting CMP-Sia, can alsodetect the precursor sialic acid, thus being useful for confirmingcellular synthesis of either or both of these compounds.

Methods for In Vivo Transfer of Sialic Acid to N-Glycans UsingSialyltrasferase

The present invention provides a method for producing a sialylatedglycoprotein in a recombinant non-human eukaryotic host cell. In oneembodiment, the non-human eukaryotic host cell does not normally displaysialyltransferase activity.

In one embodiment, said method comprises introducing into the host anucleic acid encoding a sialyltransferase enzyme. The enzyme havingsialyltransferase activity can be any enzyme having sialyltransferaseactivity including, but not limited to, any α-2,3 ST, any α-2,6 ST,human ST6Gal (GenBank AN: NM_(—)00302) or a homologue thereof, humanST3Gal (GenBank AN: L23767) or a homologue thereof, or Xenopus ST3Gal(GenBank AN: CAF22058) or a homologue thereof.

As used herein, a homologue of human ST6Gal refers to a nucleic acidhaving significant alignment with human ST6Gal (NM_(—)003032).Homologues of human ST6Gal include the enzymes identified by GenBankAccession Nos.: AAH40009.1, CAF29492.1, CAI29584.1, CAA38246.1,CAA75385.1, AAH92222.1, P13721, Q64685, NP_(—)990572.1, XP_(—)535839.1,XP_(—)517005.1, NP_(—)775324.1, XP_(—)516938.1, NP_(—)001003853.1,CAI39643.1, CAI29183.1, CAG32836.1, XP_(—)545243.1, CAF29496.1,XP_(—)416927.1, CAF29497.1, AAB22858.1, NP_(—)671738.1, CAI29184.1,AAB22859.1, BAC24793.1, BAB47506.1, CAF29493.1, XP_(—)515705.1,XP_(—)614392.1, CAF29495.1, XP_(—)236826.2, BAC87752.1, CAI29185.1,CAF97336.1, CAI39644.1, CAD54408.1, CAG05808.1, XP_(—)538436.1,BAC98272.1, XP_(—)604448.1, AAD33059.1, BAC28828.1, BAB00636.1,AAH08680.1, NP_(—)766417.1, EAA04038.2, CAH25390.1, NP_(—)726474.1, andNP_(—)523853.1.

As used herein, a homologue of human ST3Gal refers to a nucleic acidhaving significant alignment with human ST3Gal (L23767). Homologues ofhuman ST3Gal include the enzymes identified by GenBank Accession Nos.:AAA16460.1, AAM81378.1, AAH10645.1, CAH90316.1, AAM66433.1, CAF25182.1,AAK93790.1, AAM66431.1, CAA52662.1, AAM66432.1, AAF28871.1,NP_(—)976082.1, AAH11121.1, NP_(—)033204.2, AAP22942.1, CAA65076.1,NP_(—)998922.1, NP_(—)991375.1, CAB53395.1, XP_(—)522245.1, AAC14162.1,CAI29182.1, XP_(—)417860.1, AAC14163.1, BAB25732.1, BAB13940.1,CAF22058.1, NP_(—)001003854.1, CAG00953.1, CAI26289.1, XP_(—)546408.1,YP_(—)227525.1, AAH84840.1, CAF25054.1, NP_(—)989810.1, CAF25503.1,Q6KB54, NP_(—)001002883.1, AAH23312.1, CAH89922.1, CAF25178.1,AAH53179.1, AAO13870.1, AAO13869.1, AAO13867.1, AAO13866.1, AAO13861.1,AAO13859.1, NP_(—)777631.1, and NP_(—)777628.1.

As used herein, a homologue of Xenopus ST3Gal refers to a nucleic acidhaving significant alignment with Xenopus ST3Gal (CAF22058). Homologuesof Xenopus ST3Gal include the enzymes identified by GenBank AccessionNos.: CAF22058.1, CAI29182.1, XP_(—)417860.1, AAH10645.1, AAA16460.1,AAF28871.1, CAH90316.1, CAA52662.1, AAM66433.1, AAM66432.1, AAH11121.1,NP_(—)976082.1, AAP22942.1, NP_(—)033204.2, NP_(—)998922.1, CAF25182.1,NP_(—)991375.1, AAK93790.1, AAM81378.1, CAA65076.1, AAM66431.1,CAB53395.1, AAC14162.1, XP_(—)522245.1, BAB25732.1, NP_(—)001003854.1,AAC14163.1, CAI26289.1, CAG00953.1, YP_(—)227525.1, BAB13940.1,AAH84840.1, CAE51388.1, CAF25181.1, Q6KB54, NP_(—)001002882.1,AAH23312.1, NP_(—)998924.1, CAH89922.1, AAF18019.1, NP_(—)001002883.1,AAO13870.1, AAO13869.1, AAO13867.1, AAO13866.1, AAO13861.1, AAO13859.1,NP_(—)777631.1, NP_(—)777628.1, and NP_(—)777623.1.

In one embodiment, said enzyme having sialyltransferase activity isobtained using the combinatorial DNA library approach of the inventionas described herein. In one embodiment, said enzyme havingsialyltransferase activity is a fusion protein. In a preferredembodiment, said fusion protein comprises a sialyltransferase catalyticdomain and a cellular targeting signal peptide to target thesialyltransferase actity to the Golgi apparatus of the host cell. In oneembodiment, said cellular targeting signal peptide is derived from theMnn2 (leader 53) gene. More preferably, said cellular targeting signalpeptide is encoded by the first 108 bases of Mnn2 (leader 53) fromGenbank Accession Number NP_(—)009571.

Host cells may comprise a cellular pool of CMP-sialic acid that may beenhanced. In another embodiment, said host cell lacks an endogenouscellular pool of CMP-sialic acid, and may be modified to expressCMP-sialic acid. In another embodiment, said host cell has been modifiedto express one or more enzyme activities involved in the CMP-Siapathway. (See Example 16.)

In other embodiments, said host cell may be further modified to expressone or more glycosylation enzymes selected from the group consisting ofglycosyltransferases, glycosidases such as manosidases, sugartransporters and the like. In yet other embodiments, said host cell maybe modified to produce a glycoprotein comprising a terminal galactose.In other embodiments, said host cell may be modified to produce aglycoprotein comprising the Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₍₃₋₅₎GlcNAc₂. In apreferred embodiment, said host has been modified to produce a complexglycoprotein comprising Gal₂GlcNAc₂Man₃GlcNAc₂ or a hybrid glycoproteincomprising Gal₁GlcNAc₁Man₅GlcNAc₂.

In other embodiments, the invention provides a method for producing asialylated glycoprotein in a recombinant non-human eukaryotic host cellcomprising introducing into the host a nucleic acid encoding an enzymehaving sialyltransferase activity; and further comprising introducinginto the host cell one or more additional nucleic acids encoding one ormore enzymes involved in the biosynthesis or transport of CMP-Sialicacid.

In other embodiments, the host cell to be used in the methods describedherein lacks the activity of one or more enzymes selected from the groupconsisting of mannosyltransferases and phosphomannosyltransferases. Inother embodiments, the host cell to be used in the methods describedherein is an OCH1 mutant of P. pastoris.

In other embodiments, the host cell to be used in the methods describedherein is selected from the group consisting of: any Pichia sp.,including but limited to: Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae,Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichiapijperi, Pichia stiptis and Pichia methanolica; any Saccharomyces sp.including but not limited to Saccharomyces cerevisiae Hansenulapolymorpha; any Kluyveromyces sp. including but not limited toKluyveromyces lactis; Candida albicans; any Aspergillus sp. speciesincluding but not limited to Aspergillus nidulans; Aspergillus niger;and Aspergillus oryzae; Trichoderma reseei; Chrysosporium lucknowense;any Fusarium sp. including but not limited to Fusarium gramineum andFusarium venenatum; and Neurospora crassa.

The invention also provides a nucleic acid encoding a fusion proteincomprising a sialyltransferase catalytic domain functionally linked to acellular targeting signal. The invention also provides a host cellcomprising said nucleic acid sequence. The invention further provides anon-human eukaryotic host cell comprising said nucleic acid, and furtherexpressing CMP-sialic acid.

The invention further provides non-human host cell geneticallyengineered to produce a complex glycoprotein comprisingNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ or a hybrid glycoproteincomprising NANAGalGlcNAcMan₅GlcNAc₂.

The invention also provides a recombinant glycoprotein produced by anyone of the methods described herein.

Example 17 illustrates a method of the claimed invention, whereby asialylated glycoprotein is produced in vivo.

Methods for In Vivo Transfer of Sialic Acid to N-Glycans UsingTrans-Sialidase

An alternative method for obtaining sialylated proteins in non-humanhost cells that lack the CMP-Sia pathway is to use a trans-sialidase totransfer sialic acid onto an acceptable substrate glycoprotein.Protozoa, such as Trypanosoma cruzi, possess a cell-surfacetrans-sialidase which transfers sialic acid from host glycoproteins inan α 2,3 linkage to cell surface glycoproteins and glycolipids in aCMP-independent manner (see, e.g., Parodi, 1993, Schenkman, 1994). Thegene encoding trans-sialidase contains an amino terminal region withcatalytic activity and a C-terminal region with a plasma membraneanchoring domain (Pereira, 1991). T. cruzi trans-sialidase expressed inthe baculovirus-insect cell system in the presence of sialic acid donorsresults in the sialylation of exogenous galactosylated acceptors(Marchal, I. 2001). T. cruzi trans-sialidase has also been expressed inP. pastoris (Laroy, W. 2000) as a means for obtaining high amounts oftrans-sialidase for study. Thus, trans-sialidase can be used in hostcells (e.g., a fungal host such as P. pastoris) engineered herein, toproduce recombinant proteins having desirable N-glycan structures, as ameans for sialylating glycoproteins with attached oligosaccharidesbearing terminal galactose. Trans-sialidases other than that found in T.cruzi may be used. Transialidases suitable for use in the presentinvention are described for example in Colli, 1993. The disclosures ofthese references are hereby incorporated herein by reference.

Accordingly, the invention provides a method for producing a sialylatedglycoprotein in a recombinant eukaryotic host cell comprising the stepof introducing into the host a nucleic acid encoding an enzyme havingtrans-sialidase activity. The enzyme having trans-sialidase activity canbe any enzyme having trans-sialidase activity including, but not limitedto, T. cruzi trans-sialidase (e.g., GenBank Accession Nos. AJ276679,AJ002174), T. rangeli trans-sialidase (e.g., GenBank Accession No.L14943); T. brucei trans-sialidase (e.g., GenBank Accession No.XM_(—)340626), and T. carasasii trans-sialidase (e.g., GenBank AccessionNo. AY249142, AY14111), or homologues and variants thereof. In oneembodiment, said method further comprises supplementing the medium forgrowing the host cell with a sialic acid donor.

In one embodiment, said enzyme having trans-sialidase activity is afusion protein. In a preferred embodiment, said enzyme havingtrans-sialidase activity is a fusion protein comprising atrans-sialidase catalytic domain functionally linked to a cellulartargeting signal peptide. In one embodiment said cellular targetingsignal peptide is capable of targeting the trans-sialidase activity tothe cell wall of the host cell.

In another embodiment, said host cell has been engineered or selected toproduce a glycoprotein comprising a terminal galactose. In anotherembodiment, said host cell has been modified to produce a glycoproteincomprising the structure Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₍₂₋₅₎GlcNAc₂. Inpreferred embodiments, said host cell produces a complex glycoproteincomprising Gal₂GlcNAc₂Man₃GlcNAc₂ or a hybrid glycoprotein comprisingGal₁GlcNAc₁Man₅GlcNAc₂.

The invention provides a method for producing a sialylated glycoproteinin a recombinant eukaryotic host cell comprising introducing into thehost a nucleic acid encoding an enzyme having trans-sialidase activity;and further comprising introducing into the host cell one or moreadditional nucleic acids encoding one or more enzymes selected from thegroup consisting of glycosyltransferases, glycosidases such asmanossidases, sugar transporters, and enzymes involved in thebiosynthesis or transport of CMP-Sialic acid.

In one embodiment, said host cell further comprises a sialic acid donor.In one embodiment, a sialic acid donor is added to the medium used togrow the host cell. In other embodiments, one or more precursors toCMP-Sialic acid may be added to the medium. Such CMP-Sia precursorsinclude glucosamine [GlcN]; GlcNAc, UDP-GlcNAc and ManNAc. As describedearlier, in other embodiments, said host cell has been adapted tocomprise one or more sugar precursors, such as glucosamine or ManNAc,UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, or UDP-galactose.

The invention also provides a method for producing a sialylatedglycoprotein in a recombinant non-human eukaryotic host cell comprisingintroducing into a medium used for growing the host cell an enzymehaving a trans-sialidase activity and a sialic acid donor. The sialicacid donor may be CMP-Sia or the presence of sialic acid donor may beincreased by the addition of a sugar nucleotide precursor such asCMP-Sia, glucosamine, GlcNAc, UDP-GlcNAc and ManNAc. In addition, thehost cell may be adapted to comprise one or more sugar precursors, suchas glucosamine or ManNAc, UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, orUDP-galactose.

In one embodiment, the host cell to be used in the methods describedherein is selected from the group consisting of: Pichia sp., includingbut limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis and Pichia methanolica; any Saccharomyces sp includingbut not limited to Saccharomyces cerevisiae; Hansenula polymorpha; anyKluyveromyces sp. including but not limited to Kluyveromyces lactis;Candida albicans; any Aspergillus sp. including but not limited toAspergillus nidulans, Aspergillus niger and Aspergillus oryzae;Trichoderma reseei; Chrysosporium lucknowense; any Fusarium sp.including but not limited to Fusarium gramineum and Fusarium venenatum;and Neurospora crassa.

In one embodiment, the host cell to be used in the methods describedherein lacks the activity of one or more enzymes selected from the groupconsisting of mannosyltransferases and phosphomannosyltransferases. Inone embodiment, the host cell to be used in the methods described hereinis an OCH1 mutant of P. pastoris.

Introduction into a host cell of trans-sialidase activity may also beuseful even if a CMP-Sia pathway is endogenous or has been engineeredinto the host cell. Trans-sialidase activity can increase the yield ofsecreted sialic acid terminated glycoproteins by transferring sialicacid from a donor protein onto recombinantly produced glycoproteins thatdid not sialylate within the secretory pathway or which had incompletesialylation.

Whether a trans-sialidase activity is used alone, or in conjunction withan engineered CMP-Sia pathway and sialyl transferase activity,trans-sialidase may be (1) engineered to reside in the cell wall byfusion with the C-terminal domain of heat shock protein 150 (Mattila,1996) or to another cell wall anchored protein (Bgl2, CRH1 and SCW19,(Weig, 2004)); (2) fed into the culture medium; or (3) immobilized in acolumn for use in a chromatography step during protein purification.When the trans-sialidase is fed into the culture medium for growing ahost, the trans-sialidase is preferentially bound to a resin forsimplified removal during protein purification. When the trans-sialidaseis immobilized in a column, the supernatant of the host cell producing arecombinant glycoprotein is passed through the column in the presence ofa sialic acid donor to effectuate the in vitro transfer of sialic acidonto the recombiantly produced glycoprotein.

As any strategy to engineer the formation of complex N-glycans into ahost cell such as a lower eukaryote involves both the elimination aswell as the addition of particular glycosyltransferase activities, acomprehensive scheme will attempt to coordinate both requirements. Genesthat encode enzymes that are undesirable serve as potential integrationsites for genes that are desirable. For example, 1,6 mannosyltransferaseactivity is a hallmark of glycosylation in many known lower eukaryotes.The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been clonedfrom S. cerevisiae and mutations in the gene give rise to a viablephenotype with reduced mannosylation. The gene locus encoding alpha-1,6mannosyltransferase activity therefore is a prime target for theintegration of genes encoding glycosyltransferase activity. In a similarmanner, one can choose a range of other chromosomal integration sitesthat, based on a gene disruption event in that locus, are expected to:(1) improve the cells ability to glycosylate in a more human-likefashion, (2) improve the cells ability to secrete proteins, (3) reduceproteolysis of foreign proteins and (4) improve other characteristics ofthe process that facilitate purification or the fermentation processitself.

Host Cells

Although the present invention is exemplified using a P. pastoris hostorganism, it is understood by those skilled in the art that othereukaryotic host cells, including plants, algae, insects and otherspecies of yeast and fungal hosts, may be altered as described herein toproduce human-like glycoproteins. The techniques described herein foridentification and disruption of undesirable host cell glycosylationgenes, e.g. OCH1, is understood to be applicable for these and/or otherhomologous or functionally related genes in other eukaryotic host cellssuch as other yeast and fungal strains. Preferably, robust proteinproduction strains of fungal hosts that are capable of performing wellin an industrial fermentation process are selected. These strainsinclude, without limitation: any Pichia sp. including but limited to:Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichiakoclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis and Pichia methanolica; any Saccharomyces sp. includingbut not limited to Saccharomyces cerevisiae; Hansenula polymorpha; anyKluyveromyces sp. including but not limited to Kluyveromyces lactis;Candida albicans; any Aspergillus sp. including but not limited toAspergillus nidulans, Aspergillus niger, and Aspergillus oryzae;Trichoderma reseei; Chrysosporium lucknowense; any Fusarium sp.including but not limited to Fusarium gramineum and Fusarium venenatum;and Neurospora crassa.

Another aspect of the present invention thus relates to a non-humaneukaryotic host strain expressing glycoproteins comprising modifiedN-glycans that resemble those made by human-cells. Performing themethods of the invention in species other than yeast and fungal cells isthus contemplated and encompassed by this invention. It is contemplatedthat a combinatorial nucleic acid library of the present invention maybe used to select constructs that modify the glycosylation pathway inany eukaryotic host cell system. For example, the combinatoriallibraries of the invention may also be used in plants, algae andinsects, and in other eukaryotic host cells, including mammalian andhuman cells, to localize proteins, including glycosylation enzymes orcatalytic domains thereof, in a desired location along a host cellsecretory pathway. Preferably, glycosylation enzymes or catalyticdomains and the like are targeted to a subcellular location along thehost cell secretory pathway where they are capable of functioning, andpreferably, where they are designed or selected to function mostefficiently.

As described in Examples 19 and 20, plant and insect cells may beengineered to alter the glycosylation of expressed proteins using thecombinatorial library and methods of the invention. Furthermore,glycosylation in mammalian cells, including human cells, may also bemodified using the combinatorial library and methods of the invention.It may be possible, for example, to optimize a particular enzymaticactivity or to otherwise modify the relative proportions of variousN-glycans made in a mammalian host cell using the combinatorial libraryand methods of the invention.

Examples of modifications to glycosylation which can be affected using amethod according to this embodiment of the invention are: (1)engineering a eukaryotic host cell to trim mannose residues fromMan₈GlcNAc₂ to yield a Man₅GlcNAc₂ N-glycan; (2) engineering eukaryotichost cell to add an N-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂by action of GlcNAc transferase I; (3) engineering a eukaryotic hostcell to functionally express an enzyme such as an N-acetylglucosaminylTransferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II,fucosyltransferase (FT), galactosyl tranferase (GalT) or asialyltransferase (ST); (4) an engineering or CMP-Sia pathway.

The invention also provides a host cell which lacks (or lacks anefficient) endogenous CMP-Sia biosynthetic pathway and which expresses afunctional recombinant CMP-Sia biosynthetic pathway. Preferably, thehost produces a cellular pool of CMP-Sia that may be used as a donormolecule in the presence of a sialyltransferase and a glycan acceptor(e.g., Gal₂GlcNAc₂Man₃GlcNAc₂) in a sialylation reaction. Using themethods of the invention, a variety of different hosts producing CMP-Siamay be generated.

By repeating the method, increasingly complex glycosylation pathways canbe engineered into a target host, such as a lower eukaryoticmicroorganism. In one preferred embodiment, the host organism istransformed two or more times with DNA libraries including sequencesencoding glycosylation activities. Selection of desired phenotypes maybe performed after each round of transformation or alternatively afterseveral transformations have occurred. Complex glycosylation pathwayscan be rapidly engineered in this manner.

Target Glycoproteins and Glycoprotein Compositions

The methods described herein are useful for producing glycoproteins,especially glycoproteins used therapeutically in humans, as well ascompositions comprising such glycoproteins. Glycoproteins havingspecific glycoforms may be especially useful, for example, in thetargeting of therapeutic proteins. Glycoprotein compositions of thepresent invention will comprise predominantly a specific glycoform. Forexample, mannose-6-phosphate has been shown to direct proteins to thelysosome, which may be essential for the proper function of severalenzymes related to lysosomal storage disorders such as Gaucher's,Hunter's, Hurler's, Scheie's, Fabry's and Tay-Sachs disease, to mentionjust a few. Likewise, the addition of one or more sialic acid residuesto a glycan side chain may increase the lifetime of a therapeuticglycoprotein in vivo after administration. Accordingly, host cells(e.g., lower eukaryotic or mammalian) may be genetically engineered toincrease the extent of terminal sialic acid in glycoproteins expressedin the cells. Alternatively, sialic acid may be conjugated to theprotein of interest in vitro prior to administration using a sialic acidtransferase and an appropriate substrate. Changes in growth mediumcomposition may be employed in addition to the expression of enzymeactivities involved in human-like glycosylation to produce glycoproteinsmore closely resembling human forms (Weikert, 1999; Werner, 1998;Andersen, 1994; Yang, 2000). Specific glycan modifications to monoclonalantibodies (e.g. the addition of a bisecting GlcNAc) have been shown toimprove antibody dependent cell cytotoxicity (Umana, 1999), which may bedesirable for the production of antibodies or other therapeuticproteins.

Therapeutic proteins are typically administered by injection, orally,pulmonary, or other means. Examples of suitable target glycoproteinswhich may be produced according to the invention include, withoutlimitation: erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, TNFα and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,antithrombin III and thrombopoietin soluble IgE receptor α-chain, IgG,IgG fragments, IgM, interleukins such as IL-lra, urokinase, chymase,urea trypsin inhibitor, IGF-binding protein, epidermal growth factor,growth hormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, myeloid progenitor inhibitoryfactor-1, osteoprotegerin, α-1 antitrypsin, DNase II, α-proteins, AAT,rhTBP-1 (onercept, aka TNF Binding protein 1), TACI-Ig (transmembraneactivator and calcium modulator and cyclophilin ligand interactor), FSH(follicle stimulating hormone), GM-CSF, GLP-1 w/ and w/o FC (glucagonlike protein 1) IL-1 receptor agonist, sTNFr (enbrel, aka soluble TNFreceptor Fc fusion) ATIII, rhThrombin, glucocerebrosidase and CTLA4-Ig(Cytotoxic T Lymphocyte associated Antigen 4-Ig)

The following are examples which illustrate the compositions and methodsof this invention. These examples should not be construed as limiting:the examples are included for the purposes of illustration only. Forexample, while the examples illustrate embodiments of the compositionsand methods of this invention relating to complex glycoproteins, oneskilled in the art will recognize that the methods and compositionsillustrated in the examples may be adapted to compositions and methodsrelating to hybrid glycoproteins, as described earlier herein.

Example 1 Cloning and Disruption of the OCH1 Gene in P. pastoria

Generation of an OCH1 Mutant of P. pastoris

A 1215 bp ORF of the P. pastoris OCH1 gene encoding a putative α-1,6mannosyltransferase was amplified from P. pastoris genomic DNA (strainX-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO:7) and5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO:8) which were designed based onthe P. pastoris OCH1 sequence (Japanese Patent Application PublicationNo. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream ofthe ORF of the OCH1 gene were amplified from a P. pastoris genomic DNAlibrary (Boehm, 1999) using the internal oligonucleotides5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO:9) in the OCH1 gene, and5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO:10) and5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO:11) oligonucleotides in thebackbone of the library bearing plasmid lambda ZAP II (Stratagene, LaJolla, Calif.). The resulting 5075 bp fragment was cloned into thepCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9.

After assembling a gene knockout construct that substituted the OCH1reading frame with a HIS4 resistance gene, P. pastoris was transformedand colonies were screened for temperature sensitivity at 37° C. OCH1mutants of S. cerevisiae are temperature sensitive and are slow growersat elevated temperatures. One can thus identify functional homologs ofOCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiaewith a P. pastoris DNA or cDNA library. About 20 temperature sensitivestrains were further subjected to a colony PCR screen to identifycolonies with a deleted och1 gene. Several och1 deletions were obtained.

The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5 kbdown stream sequence of OCH1 gene cassette carrying Pichia HIS4 gene,was transformed into P. pastoris BK1 [GS115 (his4 Invitrogen Corp., SanDiego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knockout the wild-type OCH1 gene. The initial screening of transformants wasperformed using histidine drop-out medium followed by replica plating toselect the temperature sensitive colonies. Twenty out of two hundredhistidine-positive colonies showed a temperature sensitive phenotype at37° C. To exclude random integration of pBK9.1 into the Pichia genome,the 20 temperature-sensitive isolates were subjected to colony PCR usingprimers specific to the upstream sequence of the integration site and toHIS4 ORF. Two out of twenty colonies were och1 defective and furtheranalyzed using a Southern blot and a Western blot indicating thefunctional och1 disruption by the och1 knock-out construct. Genomic DNAwere digested using two separate restriction enzymes BglII and ClaI toconfirm the och1 knock-out and to confirm integration at the openreading frame. The Western Blot showed och1 mutants lacking a discreteband produced in the GS115 wild type at 46.2 kDa.

Example 2 Engineering of P. pastoris with α-1,2-Mannosidase to ProduceMan₅GlcNAc₂-Containing IFN-β Precursors

An α-1,2-mannosidase is required for the trimming of Man₈GlcNAc₂ toyield Man₅GlcNAc₂, an essential intermediate for complex N-glycanformation. While the production of a Man₅GlcNAc₂ precursor is essential,it is not necessarily sufficient for the production of hybrid andcomplex glycans because the specific isomer of Man₅GlcNAc₂ may or maynot be a substrate for GnTI. An och1 mutant of P. pastoris is engineeredto express secreted human interferon-β under the control of an aoxpromoter. A DNA library is constructed by the in-frame ligation of thecatalytic domain of human mannosidase IB (an α-1,2-mannosidase) with asub-library including sequences encoding early Golgi and ER localizationpeptides. The DNA library is then transformed into the host organism,resulting in a genetically mixed population wherein individualtransformants each express interferon-β as well as a syntheticmannosidase gene from the library. Individual transformant colonies arecultured and the production of interferon is induced by addition ofmethanol. Under these conditions, over 90% of the secreted protein isglycosylated interferon-β.

Supernatants are purified to remove salts and low-molecular weightcontaminants by C₁₈ silica reversed-phase chromatography. Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce interferon-β including N-glycans of thestructure Man₅GlcNAc₂, which has a reduced molecular mass compared tothe interferon-β of the parent strain. The purified interferon-β isanalyzed by MALDI-TOF mass spectroscopy and colonies expressing thedesired form of interferon-β are identified.

Example 3 Generation of an Och1 Mutant Strain Expressing anα-1,2-Mannosidase, GnTI and GnTII for Production of a Human-LikeGlycoprotein

The 1215 bp open reading frame of the P. pastoris OCH1 gene as well as2685 bp upstream and 1175 bp downstream was amplified by PCR (see alsoWO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen) anddesignated pBK9. To create an och1 knockout strain containing multipleauxotrophic markers, 100 μg of pJN329, a plasmid containing anoch1::URA3 mutant allele flanked with SfiI restriction sites wasdigested with SfiI and used to transform P. pastoris strain JC308(Cereghino et al. Gene 263 (2001) 159-169) by electroporation. Followingincubation on defined medium lacking uracil for 10 days at roomtemperature, 1000 colonies were picked and re-streaked. URA⁺ clones thatwere unable to grow at 37° C., but grew at room temperature, weresubjected to colony PCR to test for the correct integration of theoch1:: URA3 mutant allele. One clone that exhibited the expected PCRpattern was designated YJN153. The Kringle 3 domain of human plasminogen(K3) was used as a model protein. A Neo^(R) marked plasmid containingthe K3 gene was transformed into strain YJN153 and a resulting strain,expressing K3, was named BK64-1.

Plasmid pPB103, containing the Kluyveromyces lactis MNN2-2 gene whichencodes a Golgi UDP-N-acetylglucosamine transporter was constructed bycloning a blunt BglII-HindIII fragment from vector pDL02 (Abeijon et al.(1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) into BglII and BamHIdigested and blunt ended pBLADE-SX containing the P. pastoris ADE1 gene(Cereghino et al. (2001) Gene 263:159-169). This plasmid was linearizedwith EcoNI and transformed into strain BK64-1 by electroporation and onestrain confirmed to contain the MNN2-2 by PCR analysis was named PBP1.

A library of mannosidase constructs was generated, comprising in-framefusions of the leader domains of several type I or type II membraneproteins from S. cerevisiae and P. pastoris fused with the catalyticdomains of several α-1,2-mannosidase genes from human, mouse, fly, wormand yeast sources (see, e.g., WO02/00879, incorporated herein byreference). This library was created in a P. pastoris HIS4 integrationvector and screened by linearizing with SalI, transforming byelectroporation into strain PBP1, and analyzing the glycans releasedfrom the K3 reporter protein. One active construct chosen was a chimeraof the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fusedwith a N-terminal deletion of the mouse α-1,2-mannosidase IA gene (FIG.3), which was missing the 187 nucleotides. A P. pastoris strainexpressing this construct was named PBP2.

A library of GnTI constructs was generated, comprising in-frame fusionsof the same leader library with the catalytic domains of GnTI genes fromhuman, worm, frog and fly sources (WO 02/00879). This library wascreated in a P. pastoris ARG4 integration vector and screened bylinearizing with AatII, transforming by electroporation into strainPBP2, and analyzing the glycans released from K3. One active constructchosen was a chimera of the first 120 bp of the S. cerevisiae MNN9 genefused to a deletion of the human GnTI gene, which was missing the first154 bp. A P. pastoris strain expressing this construct was named PBP3.

A library of GnTII constructs was generated, which comprised in-framefusions of the leader library with the catalytic domains of GnTII genesfrom human and rat sources (WO 02/00879). This library was created in aP. pastoris integration vector containing the NST^(R) gene conferringresistance to the drug nourseothricin. The library plasmids werelinearized with EcoRI, transformed into strain RDP27 by electroporation,and the resulting strains were screened by analysis of the releasedglycans from purified K3.

Materials for the Following Reactions

MOPS, sodium cacodylate, manganese chloride, UDP-galactose andCMP-N-acetylneuraminic acid were from Sigma. Trifluoroacetic acid (TFA)was from Sigma/Aldrich, Saint Louis, Mo. Recombinant ratα2,6-sialyltransferase from Spodoptera frugiperda andβ1,4-galactosyltransferase from bovine milk were from Calbiochem (SanDiego, Calif.). Protein N-glycosidase F, mannosidases, andoligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearlresin was from TosoHaas. Metal chelating “HisBind” resin was fromNovagen (Madison, Wis.). 96-well lysate-clearing plates were fromPromega (Madison, Wis.). Protein-binding 96-well plates were fromMillipore (Bedford, Mass.). Salts and buffering agents were from Sigma(St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.).

Protein Purification

Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle3 was purified from expression media using a C-terminal hexa-histidinetag. The robotic purification is an adaptation of the protocol providedby Novagen for their HisBind resin. Briefly, a 150 uL (μL) settledvolume of resin is poured into the wells of a 96-well lysate-bindingplate, washed with 3 volumes of water and charged with 5 volumes of 50mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole,0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media isdiluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) andloaded onto the columns. After draining, the columns are washed with 10volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole,0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HClpH7.9). The eluted glycoproteins are evaporated to dryness bylyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by amodification of a previously reported method (Papac, 1998). The wells ofa 96-well MultiScreen IP (Immobilon-P membrane) plate (Millipore) arewetted with 100 uL of methanol, washed with 3×150 uL of water and 50 uLof RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining withgentle vacuum after each addition. The dried protein samples aredissolved in 30 uL of RCM buffer and transferred to the wells containing10 uL of RCM buffer. The wells are drained and washed twice with RCMbuffer. The proteins are reduced by addition of 60 uL of 0.1M DTT in RCMbuffer for 1 hr at 37° C. The wells are washed three times with 300 uLof water and carboxymethylated by addition of 60 uL of 0.1M iodoaceticacid for 30 min in the dark at room temperature. The wells are againwashed three times with water and the membranes blocked by the additionof 100 uL of 1% PVP 360 in water for 1 hr at room temperature. The wellsare drained and washed three times with 300 uL of water anddeglycosylated by the addition of 30 uL of 10 mM NH₄HCO₃ pH 8.3containing one milliunit of N-glycanase (Glyko). After 16 hours at 37°C., the solution containing the glycans was removed by centrifugationand evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

Molecular weights of the glycans were determined using a Voyager DE PROlinear MALDI-TOF (Applied Biosciences) mass spectrometer using delayedextraction. The dried glycans from each well were dissolved in 15 uL ofwater and 0.5 uL spotted on stainless steel sample plates and mixed with0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowedto dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm)with a 4 ns pulse time. The instrument was operated in the delayedextraction mode with a 125 ns delay and an accelerating voltage of 20kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, theinternal pressure was less than 5×10-7 torr, and the low mass gate was875 Da. Spectra were generated from the sum of 100-200 laser pulses andacquired with a 2 GHz digitizer. Man₅GlcNAc₂ oligosaccharide was used asan external molecular weight standard. All spectra were generated withthe instrument in the positive ion mode. The estimated mass accuracy ofthe spectra was 0.5%.

Example 4 Engineering a Strain to Produce GalactosyltransferaseGalactosyltransferase Reaction

Approximately 2 mg of protein (r-K3:hPg [PBP6-5]) was purified bynickel-affinity chromatography, extensively dialyzed against 0.1% TFA,and lyophilized to dryness. The protein was redissolved in 150 μL of 50mM MOPS, 20 mM MnCl2, pH7.4. After addition of 32.5 μg (533 nmol) ofUDP-galactose and 4 mU of β 1,4-galactosyltransferase, the sample wasincubated at 37° C. for 18 hours. The samples were then dialyzed against0.1% TFA for analysis by MALDI-TOF mass spectrometry.

The spectrum of the protein reacted with galactosyltransferase showed anincrease in mass consistent with the addition of two galactose moietieswhen compared with the spectrum of a similar protein sample incubatedwithout enzyme. Protein samples were next reduced, carboxymethylated anddeglycosylated with PNGase F. The recovered N-glycans were analyzed byMALDI-TOF mass spectrometry. The mass of the predominant glycan from thegalactosyltransferase reacted protein was greater than that of thecontrol glycan by a mass consistent with the addition of two galactosemoieties (325.4 Da).

Bobrowicz et al. (2004), which is incorporated by reference herein,discloses engineering a strain of P. pastoris capable of producingglycoproteins containing terminal galactose. This strain expressed a β1,4GalT, a UDP galactose transporter and UDP-galactose-4-epimerase usingthe methods of the invention, as disclosed herein. See also thedisclosure of Davidson, U.S. Ser. No. 11/108,088, filed on Apr. 15,2005, the disclosure of which is hereby incorporated by referenceherein.

Example 5 Engineering a Strain to Express Functional and ActiveMannosidase II

To generate a human-like glycoform, a microorganism is engineered toexpress a mannosidase II enzyme which removes the two remaining terminalmannoses from the structure GlcNAcMan₅GlcNAc₂ (see FIG. 1B). A DNAlibrary including sequences encoding cis and medial Golgi localizationsignals is fused in-frame to a library encoding mannosidase II catalyticdomains. The host organism is a strain, e.g. a yeast, that is deficientin hypermannosylation (e.g. an och1 mutant) and provides N-glycanshaving the structure GlcNAcMan₅GlcNAc₂ in the Golgi and/or ER. Aftertransformation, organisms having the desired glycosylation phenotype areselected. An in vitro assay is used in one method. The desired structureGlcNAcMan₃GlcNAc₂ (but not the undesired GlcNAcMan₅GlcNAc₂) is asubstrate for the enzyme GlcNAc Transferase II (see FIG. 1B).Accordingly, single colonies may be assayed using this enzyme in vitroin the presence of the substrate, UDP-GlcNAc. The release of UDP isdetermined either by HPLC or an enzymatic assay for UDP. Alternatively,radioactively labeled UDP-GlcNAc or MALDI-TOF may be used. See Davidsonet al., WO05/00584, the disclosure of which is hereby incorporated byreference.

The foregoing in vitro assays are conveniently performed on individualcolonies using high-throughput screening equipment. Alternatively alectin binding assay is used. In this case the reduced binding oflectins specific for terminal mannoses allows the selection oftransformants having the desired phenotype. For example, Galantusnivalis lectin binds specifically to terminal α-1,3-mannose, theconcentration of which is reduced in the presence of operativelyexpressed mannosidase II activity. In one suitable method, G. nivalislectin attached to a solid agarose support (available from SigmaChemical, St. Louis, Mo.) is used to deplete the transformed populationof cells having high levels of terminal α-1,3-mannose.

Example 6 Engineering a Strain to Express Sialyltransferase

The enzymes α2,3-sialyltransferase and α2,6-sialyltransferase addterminal sialic acid to galactose residues in nascent human N-glycans,leading to mature glycoproteins (see “α 2,3 ST; α2,6 ST” in FIG. 1B). Inhuman cells, the reactions occur in the trans Golgi or TGN. Accordingly,a DNA library is constructed by the in-frame fusion of sequencesencoding sialyltransferase catalytic domains with sequences encodingtrans Golgi or TGN localization signals (Malissard, 2000; Borsig, 1995).The host organism is a strain, e.g. a yeast, that is deficient inhypermannosylation (e.g., an och1 mutant), which provides N-glycanshaving terminal galactose residues in the late Golgi or TGN, andprovides a sufficient concentration of CMP-sialic acid in the late Golgior TGN. Following transformation, transformants having the desiredphenotype are selected, e.g., using a fluorescent antibody specific forN-glycans having a terminal sialic acid. In addition, the strains areengineered to produce the CMP-NANA precursors as described in Example16.

Example 17 further illustrates the engineering of a strain thatexpresses α2,6 ST and CMP-Sia and is able to produce glycoproteinscomprising the NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform.

Sialyltransferase Reaction

After resuspending the (galactosyltransferase reacted) (Example 4)proteins in 10 μL of 50 mM sodium cacodylate buffer pH6.0, 300 μg (488nmol) of CMP-N-acetylneuraminic acid (CMP-NANA) dissolved in 15 μL ofthe same buffer, and 50 μL (2 mU) of recombinant α-2,6 sialyltransferasewere added. After incubation at 37° C. for 15 hours, an additional 200μg of CMP-NANA and 1 mU of sialyltransferase were added. The proteinsamples were incubated for an additional 8 hours and then dialyzed andanalyzed by MALDI-TOF-MS as above. The spectrum of the glycoproteinreacted with sialyltransferase showed an increase in mass when comparedwith that of the starting material (the protein aftergalactosyltransferase reaction). The N-glycans were released andanalyzed as above. The increase in mass of the two ion-adducts of thepredominant glycan was consistent with the addition of two sialic acidresidues (580 and 583 Da).

Example 7 Engineering a Strain to Express UDP-GlcNAc Transporter

The cDNA of human Golgi UDP-GlcNAc transporter has been cloned by Ishidaand coworkers. (Ishida, N., et al. 1999 J. Biochem. 126(1): 68-77).Guillen and coworkers have cloned the canine kidney Golgi UDP-GlcNActransporter by phenotypic correction of a Kluyveromyces lactis mutantdeficient in Golgi UDP-GlcNAc transport. (Guillen, E., et al. 1998).Thus a mammalian Golgi UDP-GlcNAc transporter gene has all of thenecessary information for the protein to be expressed and targetedfunctionally to the Golgi apparatus of yeast. These or other clonedtransporter genes may be engineered into a host organism to provideUDP-GlcNAc substrates for efficient GnT reactions in the Golgi and/or ERof the host. FIG. 10B demonstrates the effect of a strain expressing aK. lactis UDP-GlcNAc transporter. In comparison to FIG. 10A, which lacksa UDP-GlcNAc transporter, the effect of adding a UDP-GlcNAc transportershows a dramatic increase in the production of GlcNAcMan₅GlcNAc₂.

Example 8 Engineering a Strain to Express GDP-Fucose Transporter

The rat liver Golgi membrane GDP-fucose transporter has been identifiedand purified by Puglielli, L. and C. B. Hirschberg 1999 J. Biol. Chem.274(50):35596-35600. The corresponding gene can be identified usingstandard techniques, such as N-terminal sequencing and Southern blottingusing a degenerate DNA probe. The intact gene is then expressed in ahost microorganism that also expresses a fucosyltransferase.

Example 9 Engineering a Strain to Express UDP-Galactose Transporter

Human UDP-galactose (UDP-Gal) transporter has been cloned and shown tobe active in S. cerevisiae. (Kainuma, M., et al. 1999 Glycobiology 9(2):133-141). A second human UDP-galactose transporter (hUGT1) has beencloned and functionally expressed in Chinese Hamster Ovary Cells. Aoki,K., et al. 1999 J. Biochem. 126(5): 940-950. Likewise, Segawa andcoworkers have cloned a UDP-galactose transporter fromSchizosaccharomyces pombe (Segawa, H., et al. 1999 Febs Letters 451(3):295-298). These or other sequences encoding UDP-galactose transporteractivities may be introduced into a host cell directly or may be used asa component of a sub-library of the invention to engineer a strainhaving increased UDP-galactose transporter activity.

Example 10 Engineering a Strain to Express CMP-Sialic Acid Transporter

Human CMP-sialic acid transporter (hCST) has been cloned and expressedin Lec 8 CHO cells by Aoki and coworkers (1999). Molecular cloning ofthe hamster CMP-sialic acid transporter has also been achieved(Eckhardt, 1997). The functional expression of the murine CMP-sialicacid transporter was achieved in Saccharomyces cerevisiae by Berninsone,1997. These or other sequences encoding CMP-sialic acid transporteractivities may be introduced into a host cell directly or may be used asa component of a sub-library of the invention to engineer a strainhaving increased CMP-sialic acid transporter activity.

Example 11 Engineering of P. pastoris to Produce Man₅GlcNA₂ as thePredominant N-Glycan Structure Using a Combinatorial DNA Library

An och1 mutant of P. pastoris (see Examples 1 and 3) was engineered toexpress and secrete proteins such as the kringle 3 domain of humanplasminogen (K3) under the control of the inducible AOXI promoter. TheKringle 3 domain of human plasminogen (K3) was used as a model protein.A DNA fragment encoding the K3 was amplified using Pfu turbo polymerase(Strategene, La Jolla, Calif.) and cloned into EcoRI and XbaI sites ofpPICZaA (Invitrogen, Carlsbad, Calif.), resulting in a C-terminal 6-Histag. In order to improve the N-linked glycosylation efficiency of K3(Hayes, 1975), Pro₄₆ was replaced with Ser₄₆ using site-directedmutagenesis. The resulting plasmid was designated pBK64. The correctsequence of the PCR construct was confirmed by DNA sequencing.

A combinatorial DNA library was constructed by the in-frame ligation ofmurine α-1,2-mannosidase IB (Genbank: 6678787) and IA (Genbank: 6754619)catalytic domains with a sub-library including sequences encoding Cop IIvesicle, ER, and early Golgi localization peptides according to Table 6.The combined DNA library was used to generate individual fusionconstructs, which were then transformed into the K3 expressing hostorganism, resulting in a genetically mixed population wherein individualtransformants each express K3 as well as a localizationsignal/mannosidase fusion gene from the library. Individualtransformants were cultured and the production of K3 was induced bytransfer to a methanol containing medium. Under these conditions, after24 hours of induction, over 90% of the protein in the medium was K3. TheK3 reporter protein was purified from the supernatant to remove saltsand low-molecular weight contaminants by Ni-affinity chromatography.Following affinity purification, the protein was desalted by sizeexclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.)and either directly subjected to MALDI-TOF analysis described below orthe N-glycans were removed by PNGase digestion as described below(Release of N-glycans) and subjected to MALDI-TOF analysis (Miele,1997).

Following this approach, a diverse set of transformants were obtained;some showed no modification of the N-glycans compared to the och1knockout strain; and others showed a high degree of mannose trimming(FIG. 5D, 5E). Desired transformants expressing appropriately targeted,active α-1,2-mannosidase produced K3 with N-glycans of the structureMan₅GlcNAc₂. This confers a reduced molecular mass to the glycoproteincompared to the K3 of the parent och1 deletion strain, a differencewhich was readily detected by MALDI-TOF mass spectrometry (FIG. 5).Table 7 indicates the relative Man₅GlcNAc₂ production levels.

TABLE 7 A Representative Combinatorial DNA Library OfSequences/Catalytic Domains Localization Exhibiting Relative Levels OfMan₅GlcNAc₂ Production. Catalytic Targeting peptide sequences DomainsMNS1(s) MNS1(m) MNS1(1) SEC12(s) SEC12(m) Mouse FB4 FB5 FB6 FB7 FB8mannosidase ++ + − ++ ++++ 1A Δ187 Mouse GB4 GB5 GB6 GB7 GB8 mannosidase++ + + ++ + 1B Δ58 Mouse GC4 GC5 GC6 GC7 GC8 mannosidase − +++ + + + 1BΔ99 Mouse GD4 GD5 GD6 GD7 GD8 mannosidase − − − + + 1B Δ170

TABLE 8 Another Combinatorial DNA Library Of Localization ExhibitingSequences/Catalytic Domains Relative Levels Of Man₅GlcNAc₂ Production.Catalytic Targeting peptide sequences Domains VAN1(s) VAN1(m) VAN1(l)MNN10(s) MNN10(m) MNN10(l) C. elegans BC18-5 BC19 BC20 BC27 BC28 BC29mannosidase +++++ ++++ +++ +++++ +++++ +++ 1B Δ80 C. elegans BB18 BB19BB20 BB18 BB19 BB20 mannosidase +++++ +++++ ++++ +++++ +++++ ++++ 1B Δ31

Targeting peptides were selected from MNS I (SwissProt P32906) in S.cerevisiae (long, medium and short) (see supra Nucleic Acid Libraries;Combinatorial DNA Library of Fusion Constructs) and SEC12 (SwissProtP11655) in S. cerevisiae (988-1140 nucleotides: short) and (988-1296:medium). Although majority of the targeting peptide sequences wereN-terminal deletions, some targeting peptide sequences, such as SEC12were C-terminal deletions. Catalytic domains used in this experimentwere selected from mouse mannosidase 1A with a 187 amino acid N-terminaldeletion; and mouse mannosidase 1B with a 58, 99 and 170 amino aciddeletion. The number of (+)s, as used herein, indicates the relativelevels of Man₅GlcNA₂ production. The notation (−) indicates no apparentproduction of Man₅GlcNA₂. The notation (+) indicates less than 10%production of Man₅GlcNA₂. The notation (++) indicates about 10-20%production of Man₅GlcNA₂. The notation with (+++) indicates about 20-40%production of Man₅GlcNA₂. The notation with (++++) indicates about 50%production of Man₅GlcNA₂. The notation with (+++++) indicates greaterthan 50% production of Man₅GlcNA₂.

Table 9 shows relative amount of Man₅GlcNAc₂ on secreted K3. Six hundredand eight (608) different strains of P. pastoris, Δoch1 were generatedby transforming them with a single construct of a combinatorial geneticlibrary that was generated by fusing nineteen (19) α-1,2 mannosidasecatalytic domains to thirty-two (32) fungal ER, and cis-Golgi leaders.

TABLE 9 Amount of Man₅GlcNAc₂ on secreted Number of K3 (% of totalglycans) constructs (%) N.D.*  19 ( 3.1)  0-10% 341 (56.1) 10-20%  50(8.2) 20-40&  75 (12.3) 40-60%  72 (11.8) More than 60%  51 (8.4) †Total 608 (100) *Several fusion constructs were not tested because thecorresponding plasmids could not be propagated in E.coli prior totransformation into P.pastoris. † Clones with the highest degree ofMan₅GlcNAc₂ trimming (30/51) were further analyzed for mannosidaseactivity in the supernatant of the medium. The majority (28/30)displayed detectable mannosidase activity in the supernatant (e.g. FIG.4B).

Only two constructs displayed high Man₅GlcNAc₂ levels, while lackingmannosidase activity in the medium (e.g. FIG. 4C).

Table 7 shows two constructs pFB8 and pGC5, among others, displayingMan₅GlcNA₂. Table 8 shows a more preferred construct, pBC18-5, a S.cerevisiae VAN1(s) targeting peptide sequence (from SwissProt 23642)ligated in-frame to a C. elegans mannosidase IB (Genbank AN: CAA98114)80 amino acid N-terminal deletion (Saccharomyces Van1 (s)/C. elegansmannosidase IB Δ80). This fusion construct also produces a predominantMan₅GlcNA₂ structure, as shown in FIG. 5E. This construct was shown toproduce greater than 50% Man₅GlcNA₂ (+++++).

Generation of a Combinatorial Localization/Mannosidase Library:

Generating a combinatorial DNA library of α-1,2-mannosidase catalyticdomains fused to targeting peptides required the amplification ofmannosidase domains with varying lengths of N-terminal deletions from anumber of organisms. To approach this goal, the full length open readingframes (ORFs) of α-1,2-mannosidases were PCR amplified from either cDNAor genomic DNA obtained from the following sources: Homo sapiens, Musmusculus, Drosophila melanogaster, Caenorhabditis elegans, Aspergillusnidulans and Penicillium citrinum. In each case, DNA was incubated inthe presence of oligonucleotide primers specific for the desiredmannosidase sequence in addition to reagents required to perform the PCRreaction. For example, to amplify the ORF of the M. musculusα-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC(SEQ ID NO:12) and the 3′-primerTCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO:13) were incubatedin the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) andamplified under the conditions recommended by Stratagene using thecycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68°C. for 30 sec, 72° C. for 3 min (30 cycles). Following amplification theDNA sequence encoding the ORF was incubated at 72° C. for 5 min with 1UTaq DNA polymerase (Promega, Madison, Wis.) prior to ligation intopCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP10chemically competent E. coli, as recommended by Invitrogen. The clonedPCR product was confirmed by ABI sequencing using primers specific forthe mannosidase ORF.

To generate the desired N-terminal truncations of each mannosidase, thecomplete ORF of each mannosidase was used as the template in asubsequent round of PCR reactions wherein the annealing position of the5′-primer was specific to the 5′-terminus of the desired truncation andthe 3′-primer remained specific for the original 3′-terminus of the ORF.To facilitate subcloning of the truncated mannosidase fragment into theyeast expression vector, pJN347 (FIG. 2C) AscI and PacI restrictionsites were engineered onto each truncation product, at the 5′- and3′-termini respectively. The number and position of the N-terminaltruncations generated for each mannosidase ORF depended on the positionof the transmembrane (TM) region in relation to the catalytic domain(CD). For instance, if the stem region located between the TM and CD wasless than 150 bp, then only one truncation for that protein wasgenerated. If, however, the stem region was longer than 150 bp theneither one or two more truncations were generated depending on thelength of the stem region.

An example of how truncations for the M. musculus mannosidase IA(Genbank: 6678787) were generated is described herein, with a similarapproach being used for the other mannosidases. FIG. 3 illustrates theORF of the M. musculus α-1,2-mannosidase IA with the predictedtransmembrane and catalytic domains being highlighted in bold. Based onthis structure, three 5′-primers were designed (annealing positionsunderlined in FIG. 3) to generate the Δ65-, Δ105- and Δ187-N-terminaldeletions. Using the Δ65 N-terminal deletion as an example the 5′-primerused was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′ (SEQ IDNO:14) (with the AscI restriction site highlighted in bold) inconjunction with the 3′-primer5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO:15)(with the PacI restriction site highlighted in bold). Both of theseprimers were used to amplify a 1561 bp fragment under the conditionsoutlined above for amplifying the full length M. musculus mannosidase 1AORF. Furthermore, like the product obtained for the full length ORF, thetruncated product was also incubated with Taq DNA polymerase, ligatedinto pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP10and ABI sequenced. After having amplified and confirmed the sequence ofthe truncated mannosidase fragment, the resulting plasmid,pCR2.1-Δ65mMannIA, was digested with AscI and PacI in New EnglandBiolabs buffer #4 (Beverly, Mass.) for 16 h at 37° C. In parallel, thepJN347 (FIG. 2C) was digested with the same enzymes and incubated asdescribed above. Post-digestion, both the pJN347 (FIG. 2C) back-bone andthe truncated catalytic domain were gel extracted and ligated using theQuick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommendedby the manufacturers, and transformed into chemically competent DH5acells (Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm thegeneration of the pJN347-mouse Mannosidase IAΔ65 construct.

Having generated a library of truncated α-1,2-mannosidase catalyticdomains in the yeast expression vector pJN347 (FIG. 2C) the remainingstep in generating the targeting peptide/catalytic domain library was toclone in-frame the targeting peptide sequences (FIG. 2). Both thepJN347-mannosidase constructs (FIG. 2D) and the pCR2.1TOPO-targetingpeptide constructs (FIG. 2B) such as were incubated overnight at 37° C.in New England Biolabs buffer #4 in the presence of the restrictionenzymes NotI and AscI. Following digestion, both the pJN347-mannosidaseback-bone and the targeting peptide regions were gel-extracted andligated using the Quick Ligation Kit (New England Biolabs, Beverly,Mass.), as recommended by the manufacturers, and transformed intochemically competent DH5a cells (Invitrogen, Carlsbad, Calif.).Subsequently, the pJN347-targeting peptide/mannosidase constructs wereABI sequenced to confirm that the generated fusions were in-frame. Theestimated size of the final targeting peptide/alpha-1,2-mannosidaselibrary contains over 1300 constructs generated by the approachdescribed above. FIG. 2 illustrates construction of the combinatorialDNA library.

Engineering a P. pastoris OCH1 Knock-Out Strain with MultipleAuxotrophic Markers.

The first step in plasmid construction involved creating a set ofuniversal plasmids containing DNA regions of the KEX1 gene of P.pastoris (Boehm et al. Yeast 1999 May; 15(7):563-72) as space holdersfor the 5′ and 3′ regions of the genes to be knocked out. The plasmidsalso contained the S. cerevisiae Ura-blaster (Alani et al., Genetics116, 541-545. 1987) as a space holder for the auxotrophic markers, andan expression cassette with a multiple cloning site for insertion of aforeign gene. A 0.9-kb fragment of the P. pastoris KEX1-5′ region wasamplified by PCR using primersGGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCAG A (SEQ ID NO:16) andGCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACCGC TACTA (SEQ ID NO:17)and P. pastoris genomic DNA as a template and cloned into the SacI, SalIsites of pUC19 (New England Biolabs, Beverly, Mass.). The resultingplasmid was cut with BamHI and SalI, and a 0.8-kb fragment of theKEX1-3′ region that had been amplified using primersCGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCCAC ATGG (SEQ ID NO:18)and GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGG ATGACTCTTTTC (SEQ IDNO:19) was cloned into the open sites creating pJN262. This plasmid wascut with BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani etal. 1987) was inserted in both possible orientations resulting inplasmids pJN263 (FIG. 4A) and pJN284 (FIG. 4B).

An expression cassette was created with NotI and PacI as cloning sites.The GAPDH promoter of P. pastoris was amplified using primersCGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT (SEQ ID NO:20) andGGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA (SEQ IDNO:21) and plasmid pGAPZ-A (Invitrogen) as template and cloned into theBamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) (FIG.4B). The resulting plasmid was cut with SpeI and SphI and the CYC1transcriptional terminator region (“TT”) that had been amplified usingprimers CCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCA CGGGTCCCA (SEQ IDNO:22) and GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTCCC (SEQ ID NO:23) and plasmid pPICZ-A (Invitrogen) as atemplate was cloned into the open sites creating pJN261 (FIG. 4B).

A knockout plasmid for the P. pastoris OCH1 gene was created bydigesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of theOCH1-5′ region, which had been amplified using the primersGAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATT CAAACACAAGGCATTGC (SEQ IDNO:24) and CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC (SEQ ID NO:25)and P. pastoris genomic DNA as a template, was cloned into the opensites (FIG. 4C). The resulting plasmid was cut with EcoRI and PmeI and a1.0-kb DNA fragment of the OCH1-3′ region that had been generated usingthe primers TGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAG ATTAGAGAATG (SEQID NO:26) and AAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTT TTCATC (SEQID NO:27) was inserted to generate pJN298 (FIG. 4C). To allow for thepossibility to simultaneously use the plasmid to introduce a new gene,the BamHI expression cassette of pJN261 (FIG. 4B) was cloned into theunique BamHI site of pJN298 (FIG. 4C) to create pJN299 (FIG. 4E).

The P. pastoris Ura3-blaster cassette was constructed using a similarstrategy as described in Lu. P., et al. 1998 (Cloning and disruption ofthe β-isopropylmalate dehydrogenase gene (Leu2) of Pichia stipidis withURA3 and recovery of the double auxotroph. Appl. Microbiol. Biotechnol.49, 141-146.) A 2.0-kb PstI, SpeI fragment of P. pastoris URA3 wasinserted into the PstI, XbaI sites of pUC19 (New England Biolabs,Beverly, Mass.) to create pJN306 (FIG. 4D). Then a 0.7-kb SacI, PvuIIDNA fragment of the lacZ open reading frame was cloned into the SacI,SmaI sites to yield pJN308 (FIG. 4D). Following digestion of pJN308(FIG. 4D) with PstI, and treatment with T4 DNA polymerase, theSacI-PvuII fragment from lacZ that had been blunt-ended with T4 DNApolymerase was inserted generating pJN315 (FIG. 4D). The lacZ/URA3cassette was released by digestion with SacI and SphI, blunt ended withT4 DNA polymerase and cloned into the backbone of pJN299 that had beendigested with PmeI and AflII and blunt ended with T4 DNA polymerase. Theresulting plasmid was named pJN329 (FIG. 4E).

A HIS4 marked expression plasmid was created by cutting pJN261 (FIG. 4F)with EcoICRI (FIG. 4F). A 2.7 kb fragment of the Pichia pastoris HIS4gene that had been amplified using the primersGCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATAA AAATACGG (SEQ IDNO:28) and GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTAAA ATCTCTAA(SEQ ID NO:29) cut with NgoMIV and SwaI and then blunt-ended using T4DNA polymerase, was then ligated into the open site. This plasmid wasnamed pJN337 (FIG. 4F). To construct a plasmid with a multiple cloningsite suitable for fusion library construction, pJN337 was cut with NotIand PacI and the two oligonucleotidesGGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT SEQ ID NO:30) andTAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC (SEQ ID NO:31), that had beenannealed in vitro were ligated into the open sites, creating pJN347(FIG. 4F).

To create an och1 knockout strain containing multiple auxotrophicmarkers, 100 μg of pJN329 was digested with SfiI and used to transformP. pastoris strain JC308 (Cereghino, 2001) by electroporation. Followingtransformation, the URA dropout plates were incubated at roomtemperature for 10 days. One thousand (1000) colonies were picked andrestreaked. All 1000 clones were then streaked onto 2 sets of URAdropout plates. One set was incubated at room temperature, whereas thesecond set was incubated at 37° C. The clones that were unable to growat 37° C., but grew at room temperature, were subjected to colony PCR totest for the correct OCHI knockout. One clone that showed the expectedPCR signal (about 4.5 kb) was designated YJN153.

Example 12 Characterization of the Combinatorial DNA Library

Positive transformants screened by colony PCR confirming integration ofthe mannosidase construct into the P. pastoris genome were subsequentlygrown at room temperature in 50 ml BMGY buffered methanol-complex mediumconsisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphatebuffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1%glycerol as a growth medium) until OD_(600 nm) 2-6 at which point theywere washed with 10 ml BMMY (buffered methanol-complex medium consistingof 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1.5% methanol as agrowth medium) media prior to induction of the reporter protein for 24hours at room temperature in 5 ml BMMY. Consequently, the reporterprotein was isolated and analyzed by mass spectrophotometry and HPLC tocharacterize its glycan structure. Using the targeting peptides in Table6, mannosidase catalytic domains localized to either the ER or the Golgishowed significant level of trimming of a glycan predominantlycontaining Man₈GlcNAc₂ to a glycan predominantly containing Man₅GlcNAc₂.This is evident when the glycan structure of the reporter glycoproteinis compared between that of P. pastoris och1 knock-out in FIGS. 5C, 6Cand the same strain transformed with M. musculus mannosidase constructsas shown in FIGS. 5D, 5E, 6D-6F. FIGS. 5 and 6 show expression ofconstructs generated from the combinatorial DNA library which showsignificant mannosidase activity in P. pastoris. Expression of pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99) (FIG. 5D, 6E) produceda protein which has approximately 30% of all glycans trimmed toMan₅GlcNAc₂, while expression of pFB8 (Saccharomyces SEC12(m)/mousemannosidase IA Δ187) (FIG. 6F) produced approximately 50% Man₅GlcNAc₂and expression of pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidaseIB Δ80) (FIG. 5E) produced 70% Man₅GlcNAc₂.

Release of N-Glycans

The glycans were released and separated from the glycoproteins by amodification of a previously reported method (Papac et al. 1998Glycobiology 8, 445-454). After the proteins were reduced andcarboxymethylated and the membranes blocked, the wells were washed threetime with water. The protein was deglycosylated by the addition of 30 μlof 10 mM NH4HCO3 pH 8.3 containing one milliunit of N-glycanase (Glyko,Novato, Calif.). After 16 hr at 37° C., the solution containing theglycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

After the N-glycans were released by PNGase digestion, they wereanalyzed by Matrix Assisted Laser Desorption Ionization Time of FlightMass Spectrometry. Molecular weights of the glycans were determinedusing a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) massspectrometer using delayed extraction. The dried glycans from each wellwere dissolved in 15 μl of water and 0.5 μl was spotted on stainlesssteel sample plates and mixed with 0.5 μl of S-DHB matrix (9 mg/ml ofdihydroxybenzoic acid, 1 mg/ml of 5-methoxysalicilic acid in 1:1water/acetonitrile 0.1% TFA) and allowed to dry. Ions were generated byirradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulsetime. The instrument was operated in the delayed extraction mode with a125 ns delay and an accelerating voltage of 20 kV. The grid voltage was93.00%, guide wire voltage was 0.1%, the internal pressure was less than5×10-7 torr, and the low mass gate was 875 Da. Spectra were generatedfrom the sum of 100-200 laser pulses and acquired with a 500 MHzdigitizer. Man₅GlcNAc₂ oligosaccharide was used as an external molecularweight standard. All spectra were generated with the instrument in thepositive ion mode.

Example 13 Trimming In Vivo by Alpha-1,2-Mannosidase

To ensure that the novel engineered strains of Example 11 in factproduced the desired Man₅GlcNAc₂ structure in vivo, cell supernatantswere tested for mannosidase activity (see FIGS. 7-9). For eachconstruct/host strain described below, HPLC was performed at 30° C. witha 4.0 mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH₂ resin(5 μm) at a flow rate of 1.0 ml/min for 40 min. In FIGS. 7 and 8,degradation of the standard Man₉GlcNAc₂ [b] was shown to occur resultingin a peak which correlates to Man₈GlcNAc₂. In FIG. 7, the Man₉GlcNAc₂[b] standard eluted at 24.61 min and Man₅GlcNAc₂ [a] eluted at 18.59min. In FIG. 8, Man₉GlcNAc₂ eluted at 21.37 min and Man₅GlcNAc₂ at 15.67min. In FIG. 9, the standard Man₈GlcNAc₂ [b] was shown to elute at 20.88min.

P. pastoris cells comprising plasmid pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600 of about10. Cells were harvested by centrifugation and transferred to BMMY toinduce the production of K3 (kringle 3 from human plasminogen) undercontrol of an AOX1 promoter. After 24 hours of induction, cells wereremoved by centrifugation to yield an essentially clear supernatant. Analiquot of the supernatant was removed for mannosidase assays and theremainder was used for the recovery of secreted soluble K3. A singlepurification step using CM-sepharose chromatography and an elutiongradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted ina 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of theK3 derived glycans is shown in FIG. 6F. The earlier removed aliquot ofthe supernatant was further tested for the presence of secretedmannosidase activity. A commercially available standard of2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB)(Glyko, Novato, Calif.) was added to: BMMY (FIG. 7A), the supernatantfrom the above aliquot (FIG. 7B), and BMMY containing 10 ng of 75 mU/mLof α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras etal., WO 02/00856 A2) (FIG. 7C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming.

P. pastoris cells comprising plasmid pGC5 (Saccharomyces MNS/(m)/mousemannosidase IB Δ99) were similarly grown and assayed. Cells were grownat room temperature in BMGY to an OD600 of about 10. Cells wereharvested by centrifugation and transferred to BMMY to induce theproduction of K3 under control of an AOX1 promoter. After 24 hours ofinduction, cells were removed by centrifugation to yield an essentiallyclear supernatant. An aliquot of the supernatant was removed formannosidase assays and the remainder was used for the recovery ofsecreted soluble K3. A single purification step using CM-sepharosechromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mMNaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG.5D. The earlier removed aliquot of the supernatant was further testedfor the presence of secreted mannosidase activity as shown in FIG. 8B. Acommercially available standard of Man9-2-AB (Glyko, Novato, Calif.)were added to: BMMY (FIG. 8A), supernatant from the above aliquot (FIG.8B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase fromTrichoderma reesei (obtained from Contreras et al., WO 02/00856 A2)(FIG. 8C). After incubation for 24 hours at room temperature, sampleswere analyzed by amino silica HPLC to determine the extent ofmannosidase trimming.

Man9-2-AB was used as a substrate and it is evident that after 24 hoursof incubation, mannosidase activity was virtually absent in thesupernatant of the pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IAΔ187) strain digest (FIG. 7B) and pGC5 (Saccharomyces MNS/(m)/mousemannosidase IB Δ99) strain digest (FIG. 8B) whereas the positive control(purified α-1,2-mannosidase from T. reesei obtained from Contreras)leads to complete conversion of Man₉GlcNAc₂ to Man₅GlcNAc₂ under thesame conditions, as shown in FIGS. 7C and 8C. This is conclusive datashowing in vivo mannosidase trimming in P. pastoris pGC5 strain; andpFB8 strain, which is distinctly different from what has been reportedto date (Contreras et al., WO 02/00856 A2).

FIG. 9 further substantiates localization and activity of themannosidase enzyme. P. pastoris comprising pBC18-5 (SaccharomycesVAN1(s)/C. elegans mannosidase IB Δ80) was grown at room temperature inBMGY to an OD600 of about 10. Cells were harvested by centrifugation andtransferred to BMMY to induce the production of K3 under control of anAOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant. An aliquot ofthe supernatant was removed for mannosidase assays and the remainder wasused for the recovery of secreted soluble K3. A single purification stepusing CM-sepharose chromatography and an elution gradient 25 mM NaAc,pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 elutingbetween 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans isshown in FIG. 5E. The earlier removed aliquot of the supernatant wasfurther tested for the presence of secreted mannosidase activity asshown in FIG. 9B. A commercially available standard of Man8-2-AB (Glyko,Novato, Calif.) was added to: BMMY (FIG. 9A), supernatant from the abovealiquot pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)(FIG. 9B), and BMMY containing media from a different fusion constructpDD28-3 (Saccharomyces MNN10(m) (from SwissProt 50108)/H. sapiensmannosidase IB Δ99) (FIG. 9C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming. FIG. 9B demonstrates intracellularmannosidase activity in comparison to a fusion construct pDD28-3(Saccharomyces MNN10(m) H. sapiens mannosidase IB Δ99) exhibiting anegative result (FIG. 9C).

Example 14 pH Optimum Assay of Engineered α-1,2-Mannosidase

P. pastoris cells comprising plasmid pBB27-2 (Saccharomyces MNN10 (s)(from SwissProt 50108)/C. elegans mannosidase IB Δ31) were grown at roomtemperature in BMGY to an OD600 of about 17. About 80 μL of these cellswere inoculated into 600 μL BMGY and were grown overnight. Subsequently,cells were harvested by centrifugation and transferred to BMMY to inducethe production of K3 (kringle 3 from human plasminogen) under control ofan AOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant (pH 6.43). Thesupernatant was removed for mannosidase pH optimum assays.Fluorescence-labeled Man₈GlcNAc₂ (0.5 μg) was added to 20 μL ofsupernatant adjusted to various pH (FIG. 11) and incubated for 8 hoursat room temperature. Following incubation the sample was analyzed byHPLC using an Econosil NH2 4.6×250 mm, 5 micron bead, amino-bound silicacolumn (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 minand the column was maintained to 30° C. After eluting isocratically (68%A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60%B) was employed over 27 min to elute the glycans (18). Solvent A(acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. Thecolumn was equilibrated with solvent (68% A:32% B) for 20 min betweenruns.

Example 15 Engineering of P. pastoris to Produce N-Glycans with theStructure GlcNAcMan₅GlcNAc₂

GlcNAc Transferase I activity is required for the maturation of complexand hybrid N-glycans (U.S. Pat. No. 5,834,251). Man₅GlcNAc₂ may only betrimmed by mannosidase II, a necessary step in the formation of humanglycoforms, after the addition of N-acetylglucosamine to the terminalα-1,3 mannose residue of the trimannose stem by GlcNAc Transferase I(Schachter, 1991 Glycobiology 1(5):453-461). Accordingly, acombinatorial DNA library was prepared including DNA fragments encodingsuitably targeted catalytic domains of GlcNAc Transferase I genes fromC. elegans and Homo sapiens; and localization sequences from GLS, MNS,SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2, MNN2, MNN5,YURI, MNN1, and MNN6 from S. cerevisiae and P. pastoris putativeα-1,2-mannosyltransferases based on the homology from S. cerevisiae: D2,D9 and J3, which are KTR homologs. Table 10 includes but does not limittargeting peptide sequences such as SEC and OCH1, from P. pastoris andK. lactis GnTI, (See Table 6 and Table 10)

TABLE 10 A Representative Combinatorial Library Of Targeting PeptideSequences/Catalytic Domain For UDP-N- Acetylglucosaminyl Transferase I(GnTI) Catalytic Targeting peptide Domains OCHI(s) OCHI(m) OCHI(1)MNN9(s) MNN9(m) Human, GnTI, PB105 PB106 PB107 PB104 N/A Δ38 Human,GnTI, NB12 NB13 NB14 NB15 NB Δ86 C.elegans, OA12 OA13 OA14 OA15 OA16GnTI, Δ88 C.elegans, PA12 PA13 PA14 PA15 PA16 GnTI, Δ35 C.elegans, PB12PB13 PB14 PB15 PB16 GnTI, Δ63 X.leavis, GnTI, QA12 QA13 QA14 QA15 QA16Δ33 X.leavis, GnTI, QB12 QB13 QB14 QB15 QB 16 Δ103

Targeting peptide sequences were selected from OCHI in P. pastoris(long, medium and short) (see Example 11) and MNN9 (SwissProt P39107) inS. cerevisiae short, and medium. Catalytic domains were selected fromhuman GnTI with a 38 and 86 amino acid N-terminal deletion, C. elegans(gly-12) GnTI with a 35 and 63 amino acid deletion as well as C. elegans(gly-14) GnTI with a 88 amino acid N-terminal deletion and X. leavisGnTI with a 33 and 103 amino acid N-terminal deletion, respectively.

A portion of the gene encoding human N-acetylglucosaminyl Transferase I(MGATI, GenBank Accession No. NM002406), lacking the first 154 bp, wasamplified by PCR using oligonucleotides5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ ID NO:32) and 5′-AGGTTAATTAAGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO:33) and vector pHG4.5 (ATCC#79003) astemplate. The resulting PCR product was cloned into pCR2.1-TOPO and thecorrect sequence was confirmed. Following digestion with AscI and PacIthe truncated GnTI was inserted into plasmid pJN346 to create pNA. Afterdigestion of pJN271 with NotI and AscI, the 120 bp insert was ligatedinto pNA to generate an in-frame fusion of the MNN9 transmembrane domainwith the GnTI, creating pNA15.

The host organism is a strain of P. pastoris that is deficient inhypermannosylation (e.g. an och1 mutant), provides the substrateUDP-GlcNAc in the Golgi and/or ER (i.e. contains a functional UDP-GlcNActransporter), and provides N-glycans of the structure Man₅GlcNAc₂ in theGolgi and/or ER (e.g. P. pastoris pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187) from above). First, P. pastoris pFB8 wastransformed with pPB103 containing the Kluyveromyces lactis MNN2-2 gene(Genbank AN: AF106080) (encoding UDP-GlcNAc transporter) cloned intoBamHI and BglII site of pBLADE-SX plasmid (Cereghino, 2001). Then theaforementioned combinatorial DNA library encoding a combination ofexogenous or endogenous GnTI/localization genes was transformed andcolonies were selected and analyzed for the presence of the GnTIconstruct by colony PCR. Our transformation and integration efficiencywas generally above 80% and PCR screening can be omitted once robusttransformation parameters have been established.

Protein Purification

K3 was purified from the medium by Ni-affinity chromatography utilizinga 96-well format on a Beckman BioMek 2000 laboratory robot. The roboticpurification is an adaptation of the protocol provided by Novagen fortheir HisBind resin. Another screening method may be performed using aspecific terminal GlcNAc binding antibody, or a lectin such as the GSIIlectin from Griffonia simplificolia, which binds terminal GlcNAc (EYLaboratories, San Mateo, Calif.). These screens can be automated byusing lectins or antibodies that have been modified with fluorescentlabels such as FITC or analyzed by MALDI-TOF.

Secreted K3 can be purified by Ni-affinity chromatography, quantifiedand equal amounts of protein can be bound to a high protein binding96-well plate. After blocking with BSA, plates can be probed with aGSII-FACS lectin and screened for maximum fluorescent response. Apreferred method of detecting the above glycosylated proteins involvesthe screening by MALDI-TOF mass spectrometry following the affinitypurification of secreted K3 from the supernatant of 96-well culturedtransformants. Transformed colonies were picked and grown to an OD600 of10 in a 2 ml, 96-well plate in BMGY at 30° C. Cells were harvested bycentrifugation, washed in BMMY and resuspended in 250 ul of BMMY.Following 24 hours of induction, cells were removed by centrifugation,the supernatant was recovered and K3 was purified from the supernatantby Ni affinity chromatography. The N-glycans were released and analyzedby MALDI-TOF delayed extraction mass spectrometry as described herein.

In summary, the methods of the invention yield strains of P. pastoristhat produce GlcNAcMan₅GlcNAc₂ in high yield, as shown in FIG. 10B. Atleast 60% of the N-glycans are GlcNAcMan₅GlcNAc₂. To date, no reportexists that describes the formation of GlcNAcMan₅GlcNAc₂ on secretedsoluble glycoproteins in any yeast. Results presented herein show thataddition of the UDP-GlcNAc transporter along with GnTI activity producesa predominant GlcNAcMan₅GlcNAc₂ structure, which is confirmed by thepeak at 1457 (m/z) (FIG. 10B).

Construction of Strain PBP-3:

The P. pastoris strain expressing K3, (Δoch1, arg-, ade-, his-) wastransformed successively with the following vectors. First, pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed inthe P. pastoris strain by electroporation. Second, pPB103 containingKluyveromyces lactis MNN2-2 gene (Genbank AN: AF106080) (encodingUDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al.Gene 263 (2001) 159-169) digested with BamHI and BglII enzymes wastransformed in the P. pastoris strain. Third, pPB104 containingSaccharomyces MNN9(s)/human GnTI Δ38 encoding gene cloned as NotI-PacIfragment into pJN336 was transformed into the P. pastoris strain.

Example 16 Engineering a CMP-Sialic Acid Biosynthetic Pathway in P.pastoris Cloning Enzymes Involved in CMP-Sialic Acid Synthesis

One method for cloning a CMP-sialic acid biosynthetic pathway into afungal host cell involves amplifying the E. coli NeuA, NeuB and NeuCgenes from E. coli genomic DNA using the polymerase chain reaction inconjunction with primer pairs specific for each open reading frame (ORF)(Table 12 below, and FIGS. 17, 16 and 15, respectively).

For cloning a mammalian CMP-sialic acid biosynthetic pathway, the mouseCMP-Sia synthase ORF (FIG. 18) was amplified from a mouse pituitary cDNAlibrary in conjunction with the primer pairs set forth in Table 12. TheGlcNAc epimerase (previously discussed in an alternate method forproducing CMP-Sia intermediates), was amplified from porcine cDNA usingPCR in conjunction with primer pairs specific for the corresponding gene(Table 12 and FIG. 20). The sialate aldolase gene (FIG. 22) wasamplified from E. coli genomic DNA using the polymerase chain reactionin conjunction with the primer pairs set forth in Table 12. The mousebifunctional UDP-N-acetylglucosamine-2-Epimerase/N-acetylmannosaminekinase gene was amplified from mouse liver using the polymerase chainreaction in conjunction with the primer pairs set forth in Table 12. Themouse N-acetylneuraminate-9-phosphate synthase gene was amplified frommouse liver using the polymerase chain reaction in conjunction with theprimer pairs set forth in Table 12. The human CMP-Sia synthase gene wasamplified from human liver using the polymerase chain reaction inconjunction with the primer pairs set forth in Table 12. In each case,the ORFs were amplified using a high-fidelity DNA polymerase enzymeunder the following thermal cycling conditions: 97° C. for 1 min, 1cycle; 97° C. for 20 sec, 60° C. for 30 sec, 72° C. for 2 min, 25cycles; 72° C. for 2 min, 1 cycle. Following DNA sequencing to confirmthe absence of mutations, each ORF is re-amplified using primerscontaining compatible restriction sites to facilitate the subcloning ofeach into suitable fungal expression vectors.

TABLE 12 Primer name Primer sequence NeuA sense 5′-ATGAGAACAAAAATTATTGCGATAATTCCAGCCC G-3′ (SEQ ID NO: 45) NeuA antisense5′-TCATTTAACAATCTCCGCTATTTCGTTTTC-3′ (SEQ ID NO: 46) NeuB sense 5′-ATGAGTAATATATATATCGTTGCTGAAATTGGTTG- 3′ (SEQ ID NO: 47) NeuB antisense5′-TTATTCCCCCTGATTTTTGAATTCGCTATG-3′ (SEQ ID NO: 48) NeuC sense 5′-ATGAAAAAAATATTATACGTAACTGGATCTAGAG- 3′ (SEQ ID NO: 49) NeuC antisense5′- CTAGTCATAACTGGTGGTACATTCCGGGATGTC-3′ (SEQ ID NO: 50) mouse CMP-Sia5′-ATGGACGCGCTGGAGAAGGGGGCCGTCACGTC- synthase sense 3′ (SEQ ID NO: 51)mouse CMP-Sia 5′- synthase antisense CTATTTTTGGCATGAGTTATTAACTTTTTCTATCAG-3′ (SEQ ID NO: 52) porcine GlcNAc 5′-ATGGAGAAGGAGCGCGAAACTCTGCAGG-3′epimerase sense (SEQ ID NO: 53) porcine GlcNAc5′-CTAGGCGAGGCGGCTCAGCAGGGCGCTC-3′ epimerase (SEQ ID NO: 54) antisenseE. coli Sialate 5′-ATGGCAACGAATTTACGTGGCGTAATGGCTG-3′ aldolase sense(SEQ ID NO: 55) E. coli Sialate 5′-TCACCCGCGCTCTTGCATCAACTGCTGGGC-3′aldolase antisense (SEQ ID NO: 56) mouse bifunctional 5′- UDP-N-ATGGAGAAGAACGGGAACAACCGAAAGCTCCG-3′ acetylglucosamine- (SEQ ID NO: 69)2-epimerase/N- acetylmannosamine kinase sense mouse bifunctional5′-CTAGTGGATCCTGCGCGTTGTGTAGTCCAG-3′ UDP-N- (SEQ ID NO: 70)acetylglucosamine- 2-epimerase/N- acetylmannosamine kinase antisensemouse Sia9P syn 5′-ATGCCGCTGGAACTGGAGCTGTGTCCCGGGC-3′ sense(SEQ ID NO: 71) mouse Sia9P syn 5′-TTAAGCCTTGATTTTCTTGCTGTGACTTTCCAC-antisense 3′ (SEQ ID NO: 72) human N- 5′- acetylneuraminicGGGAGAATGCGGCCGCCACCATGGGGCTGAGCCG acid phosphatase C GTGCGGGCGGTTTTC-3′(NANP) sense (SEQ ID NO: 73) human N- 5′- acetylneuraminicGTATAGACTGCAAAGTCAGTATGTCCACTTGATT acid phosphatase AATTAACC-3′(SEQ ID NO: 74) (NANP) antisense human CMP-Sia5′-ATGGACTCGGTGGAGAAGGGGGCCGCCACC-3′ synthase sense (SEQ ID NO: 75)human CMP-Sia 5′-CTATTTTTGGCATGAATTATTAACTTTTTCC-3′ synthase antisense(SEQ ID NO: 76)Expression of Bacterial Neu Genes in P. pastoris

The 1176 bp PCR amplified fragment of the NeuC gene was ligated into theNotI-AscI site in the yeast integration vector pJN348 (a modified pUC19vector comprising a GAPDH promoter, a NotI AscI PacI restriction sitecassette, CycII transcriptional terminator, URA3 as a positive selectionmarker) producing pSH256. Similarly, the PCR amplified fragment (1041bp) of the NeuB gene was ligated into the NotI-PacI site in the yeastintegration vector pJN335, under the control of a GAPDH promoter usingADE as a positive selection marker, producing pSH255. The 1260 bp PCRamplified fragment of the NeuA gene was ligated into the NotI-PacI sitein the yeast integration vector pJN346, under the control of a GAPDHpromoter with ARG as a positive selection marker, to produce pSH254.After transforming P. pastoris with each vector by electroporation, thecells were plated onto the corresponding drop-out agar plates tofacilitate positive selection of the newly introduced vector(s). Toconfirm the introduction of each gene, several hundred clones wererepatched onto the respective dropout plates and grown for two days at26° C. Once sufficient material had grown, each clone was screened bycolony PCR using primers specific for the introduced gene. Conditionsfor colony PCR using the polymerase ExTaq™ from Takara (Madison, Wis.),were as follows: 97° C. for 3 min, 1 cycle; 97° C. for 20 sec, 50° C.for 30 sec, 72° C. for 2 min/kb, 30 cycles; 72° C. for 10 min, 1 cycle.Subsequently, several positive clones from colony PCR were grown in abaffled flask containing 200 ml of growth media. The base composition ofgrowth media containing 2.68 g/l yeast nitrogen base, 200 mg/l biotinand 2 g/l dextrose was supplemented with appropriate amino acidsdepending on the strain used. The cells were grown in this media in thepresence or absence of 20 mM ManNAc. Following growth in the baffleflask at 30° C. for 4-6 days, the cells were pelleted and analyzed forintermediates of the sialic acid pathway, as described below in thisExample.

Expression of GlcNAc Epimerase Gene in P. pastoris

The PCR amplified fragment of the porcine GlcNAc epimerase gene isligated into the NotI-PacI site in the yeast integration vector pJN348,under the control of the GAPDH promoter using URA3 as a positiveselection marker. The P. pastoris strain producing endogenous GlcNAc istransformed with the vector carrying the GlcNAc epimerase gene fragmentand screened for transformants.

Expression of Sialate Aldolase Gene in P. pastoris

The PCR amplified fragment of the E. coli sialate aldolase gene isligated into the NotI-PacI site in the yeast integration vector pJN335,under the control of the GAPDH promoter with ADE as a positive selectionmarker, producing pSH275. The P. pastoris strain producing ManNAc istransformed with the vector carrying the sialate aldolase gene fragmentand screened for transformants.

Expression of the Gene EncodingUDP-N-AcetylGlucosamine-2-Epimerase/N-Acetylmannosamine Kinase in P.pastoris

The PCR amplified fragment of the gene encoding the mouse bifunctionalUDP-N-acetylglucosamine-2-Epimerase/N-acetylmannosamine Kinase enzymewas ligated into the NotI-PacI site in the yeast integration vectorpJN348, under the control of the GAPDH promoter using URA as a positiveselection marker, producing pSH284. The P. pastoris strain producingManNAc was transformed with the vector carrying the gene fragment andscreened for transformants.

Expression of the Gene Encoding N-Acetyl-Neuraminate-9-PhosphateSynthase in P. pastoris

The PCR amplified fragment of the mouse N-acetylneuraminate-9-phosphatesynthase gene was ligated into the NotI-PacI site in the yeastintegration vector pJN335, under the control of the GAPDH promoter withADE as a positive selection marker producing, pSH285. The P. pastorisstrain producing ManNAc-6-P was transformed with the vector carrying theabove gene fragment and screened for transformants.

Identification, Cloning and Expression of the Gene EncodingN-Acetylneuraminate-9-Phosphatase

N-acetylneuraminate-9-phosphatase activity has been detected in thecytosolic fraction of rat liver cells (Van Rinsum, 1984). We haverepeated this method and isolated a cell extract fraction containingphosphatase activity selective against NeuAc-9-P. SDS-PAGEelectrophoresis of this fraction identifies a single protein band.Subsequently, this sample was electroblotted onto a PDVF membrane, andthe N-terminal amino acid sequence was identified by Edman degradation.The sequence identified allows the generation of degenerateoligonucleotides for the 5′-terminus of the ORF of the isolated protein.Using these degenerate primers in conjunction with the AP1 primersupplied in a rat liver Marathon-ready cDNA library (Clontech) accordingto the manufacturer's instructions, a full length ORF was isolated. Thecomplete ORF was subsequently ligated into the yeast integration vectorpJN347, under the control of the GAPDH promoter using a HIS gene as apositive selection marker. The P. pastoris strain producing NeuAc-9-Pwas transformed with the vector carrying the desired gene fragment andscreened for transformants as described above in this Example.

Cloning and Expression of a CMP-Sialic Acid Synthase Gene in P. pastoris

The PCR amplified fragment of the mouse CMP-Sia synthase gene wasligated into the NotI-PacI site in the yeast integration vector pJN346under the control of the GAPDH promoter with the ARG gene as a positiveselection marker. A P. pastoris strain producing sialic acid wastransformed with the vector carrying the above gene fragment andscreened for transformants as described above in this Example. Likewise,the human CMP-Sia synthase gene (Genbank AN: AF397212) was amplified andligated into the NotI-PacI site of the yeast expression vector pJN346under the control of a GAPDH promoter with ARG as a positive selectionmarker, producing the vector pSH257. A P. pastoris strain capable ofproducing sialic acid is transformed with pSH257 by electroporation,producing a strain capable of generating CMP-Sia.

Expression of the Hybrid CMP-Sia Pathway in P. pastoris

The P. pastoris strain JC308 (Cereghino, 2001) was super-transformed orsimultaneously transformed with 20 mg of each of the vectors containingNeuC (pSH256), NeuB (pSH255) and hCMP-Sia synthase (pSH257) byelectroporation. The resulting cells were plated on minimal mediasupplemented with histidine (containing 1.34 g/l yeast nitrogen base,200 mg/l biotin, 2 g/l dextrose, 20 g/l agar and 20 mg/l L-histidine).Following incubation at 30° C. for 4 days, several hundred clones wereisolated by repatching onto minimal media plates supplemented withhistidine (see above for composition). The repatched clones were grownfor 2 days prior to performing colony PCR (as described above in thisExample) on the clones. Primers specific for NeuC, NeuB and hCMP-Siasynthase were used to confirm the presence of each ORF in thetransformed clones. Twelve clones positive for all three ORFs(designated YSH99a-1) were grown in a baffled flask containing 200 ml ofgrowth media (containing 2.68 g/l yeast nitrogen base, 200 mg/l biotin,20 mg/l L-histidine and 2 g/l dextrose). The effect of supplementing thegrowth media with ManNAc was investigated by growing the cells in thepresence or absence of 20 mM ManNAc. Following growth in the baffleflask at 30° C. for 4-6 days the cells are pelleted and analyzed for thepresence of sialic acid pathway intermediates as described below in thisExample.

Comparing the cell extracts using the assay outlined below in thisExample, the cell extracts from P. pastoris YSH99a grown withoutexogenous CMP-Sia, showed transfer of Sia onto acceptor substrates,indicating the presence of endogenous CMP-Sia production (FIG. 25). Bothmono- and di-sialylated biantennary N-glycans eluted at 20 min and 23min, their respective corresponding time. Additionally, subsequentsialidase treatment showed the removal of sialic acid (FIG. 26). Thus, ayeast strain engineered with a hybrid CMP-Sia biosynthetic pathway asdescribed above, containing the NeuC, NeuB and hCMP-Sia synthase, wasshown to be capable of generating an endogenous pool of CMP-sialic acid.

Assay for the Presence of Cytidine-5′-Monophospho-N-AcetylneuraminicAcid in Genetically Altered P. pastoris

Yeast cells were washed three times with cold PBS buffer, and suspendedin 100 mM ammonium bicarbonate pH 8.5 and kept on ice. The cells werelysed using a French pressure cell followed by sonication. Soluble cellcontents were separated from cell debris by ultracentrifugation. Icecold ethanol was added to the supernatant to a final concentration of60% and kept on ice for 15 minutes prior to removal of insolubleproteins by ultracentrifugation. The supernatant was frozen andconcentrated by lyophilization. The dried sample was resuspended inwater (ensuring pH is 8.0) and then filtered through a pre-rinsed 10,000MWCO Centricon cartridge. The filtrate was separated on a Mono Qion-exchange column according to manufacturer's instructions and theelution fractions that co-eluted with authentic CMP-sialic acid werepooled and lyophilized.

The dried filtrate was dissolved in 100 μL of 100 mM ammonium acetatepH6.5, 11 μL (5 mU) of α-2,6 sialyltransferase and 3.3 μL (12 mU) ofα-2,3 sialyltransferase were added, and 10 μL of the mixture was removedfor a negative control. Subsequently, 7 μL (1.4 μg) of2-aminobenzamide-labeled asialo-biantennary N-glycan (NA2, Glyco Inc.,San Rafael, Calif.) was added to the remaining mixture, followed by theremoval of 10 μL for a positive control. The sample and controlreactions were then incubated at 37° C. for 16 hr. 10 μL of each samplewere then separated on a GlycoSep-C anion exchange column according tomanufacturer's instructions. A separate control consisting ofapproximately 0.05 μg each of monosialylated and disialylatedbiantennary glycans was separated on the column to establish relativeretention times. The results are shown in FIGS. 23-27.

Sialidase Treatment

The incubation of bi-antennary galactosylated N-glycans with an extractfrom the P. pastoris YSH99a strain in the presence of sialyltransferasesproduced sialylated N-glycans, which were subsequently desialylated asfollows: a sialylated sample was passed through a Microcon cartridge,with 10,000 molecular weight cut-off, to remove the transferases. Thecartridge was washed twice with 100 μl of water, which was pooled withthe original eluate. Analysis of the eluate by HPLC (FIG. 26) produced aspectrum similar to the HPLC spectrum prior to the Microcon treatment.The remaining sample was lyophilized to dryness and resuspended in 25 μlof 1×NEB G1 buffer. After addition of 100 U of sialidase (New EnglandBiolabs #P0720L, Beverley, Mass.), the resuspended sample was incubatedovernight at 37° C. prior to HPLC analysis, as described previously.

Example 17 Engineering a P. pastoris Strain Capable of TransferringSialic Acid to N-Glycan

A P. pastoris Strain Capable of Transferring Sialic Acid to N-Glycans InVivo has been Created as Described in Detail Herein.

Generation of pSH321b for Expressing HumanUDP-GlcNAc-2-Epimerase/N-Acetylmannosamine Kinase (hGNE) andN-Acetylneuraminate-9-Phosphate Synthase (HsiaPsyn)

The gene sequence encoding the humanUDP-GlcNAc-2-epimerase/N-acetylmannosamine kinase (hGNE; GenbankAccession No. AJ238764) was amplified as NotI-PacI fragment from humanliver cDNA using oligonucleotide primersGGGAGAATGCGGCCGCCACCATGGAGAAGAATGGAAATAACCGAAAGCT GCG (hGNE NotI/Koz)(SEQ ID NO: 77) and CCTTAATTAACTAGTAGATCCTGCGTGTTGTGTAGTCCAGAAC (hGNEPacI rev) (SEQ ID NO: 78) with Advantage™ DNA polymerase (BDBiosciences) according to manufacturer's instructions. The conditionsused for thermocycling were as follows: 95° C. 2 min, 1 cycle; 97° C. 30sec, 60° C. 30 sec, 72° C. 5 min, 25 cycles; 72° C. 5 min. The resulting2.2 Kb fragment was cloned into pCR2.1 (Invitrogen), sequenced anddesignated pSH281. The hGNE was cloned from pSH281 into pJN348 (asdescribed in US Publication No. 2004/0230042) as a NocI-PacI fragment,producing vector pSH284. Positive clones from colony PCR were grown in abaffled flask containing 200 mL of BMGY media consisting of 2.68 g/Lyeast nitrogen base, 200 mg/L biotin and 2 g/L dextrose, supplementedwith amino acids depending on the strains.

The gene sequence encoding the human N-acetylneuraminate-9-phosphatesynthase (hSiaPsyn; Genbank Accession No. AF257466) was amplified asabove using primers GGGAGAATGCGGCCGCCACCATGCCGCTGGAGCTGGAGCTGTGTCCCG(hSiaPsyn NotI/Koz) (SEQ ID NO: 79) andCCTTAATTAATTAAGACTTGATTTTTTTGCCATGATTATCTACC (hSiaPsyn PacI rev) (SEQ IDNO: 80). The conditions used for thermocycling were 2.5 min extensioninstead of 5 min. The resulting 1.1 Kb fragment was cloned into pCR2.1(Invitrogen), sequenced and designated pSH282. The hSiaPsyn was clonedfrom pSH282 into pJN664 (as disclosed in US Publication No. 2004/230042)as a NotI-PacI fragment, giving the vector pSH302. This construct wasused as template to amplify the PMA hSiaPsyn cassette flanked by XhoIsites using the primers: GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC (pPMAfor XhoI) (SEQ ID NO: 81) and CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG(PMAtt rev XhoI) (SEQ ID NO: 82). The resulting vector was designatedpSH315.

The 2.6 Kb XhoI fragment from pSH315, containing the PMA hSiaPsyncassette, was ligated into the XhoI site of pSH284 to give the doubleexpression cassette vector pSH321b (FIG. 28).

Generation of pJN711b for Expressing Genes Involved in MakingGalactosylated Glycoproteins

The D. melanogaster gene encoding the UDP Galactose Transporter (GenbankAccession No. BAB62747) (referred to as DmUGT) was PCR amplified from aD. melanogaster cDNA library (UC Berkeley Drosophila Genome Project,ovary λ-ZAP library GM), cloned into the pCR2.1 PCR cloning vector(Invitrogen) and sequenced. Primers DmUGT-5′(5′-GGCTCGAGCGGCCGCCACCATGAATAGCATACACATGAACGCCAATACG-3′) (SEQ ID NO:83) and DmUGT-3′ (5′-CCCTCGAGTTAATTAACTAGACGCGCGGCAGCAGCTTCTCCTCATCG-3′)(SEQ ID NO: 84) were used to amplify the gene, which introduced NotI andPacI sites at the 5′ and 3′ ends, respectively. The NotI and PacI siteswere then used to subclone DmUGT fused downstream of the PpOCH1 andpromoter at the NotI/PacI sites in pRCD393 to create plasmid pSH263. SeeDavidson et al., WO05/100584A2, which is incorporated by referenceherein.

The S. pombe gene encoding UDP-galactose 4-epimerase (GenBank AccessionNo. ATCC24843) (referred to as SpGALE) was amplified from S. pombegenomic DNA using primers GALE2-L (5′ ATG ACT GGT GTT CAT GAA GGG 3′)(SEQ ID NO: 85) and GALE2-R (5′ TTA CTT ATA TGT CTT GGT ATG 3′) (SEQ IDNO: 86). The amplified product was cloned into pCR2.1 (Invitrogen) andsequenced. Sequencing revealed the presence of an intron (175 bp) at the+66 position. To eliminate the intron, upstream primer GD1 (94 bases)was designed. It has a NotI site, 66 bases upstream of the intron,followed by 20 bases preceding the intron. GD2 is the downstream primerand has a PacI site. Primers GD1 (5′ GCG GCC GCA TGA CTG GTG TTC ATG AAGGGA CTG TGT TGG TTA CTG GCG GCG CTG GTT

ATA TAG GTT CTC ATA CGT GCG TTG TTT TGT TAG AAA A 3′) (SEQ ID NO: 87)and GD2 (5′ TTA ATT AAT TAC TTA TAT GTC TTG GTA TG 3′) (SEQ ID NO: 88)were used to amplify the SpGALE intronless gene from the pCR2.1 subcloneand the product cloned again into pCR2.1 and sequenced.

The H. sapiens β-1,4-galactosyltransferase I gene (hGalTI, GenbankAccession No. AH003575) was PCR amplified from human kidney cDNA(Marathon-Ready cDNA, Clontech) using primers RCD192(5′-GCCGCGACCTGAGCCGCCTGCCCCAAC-3′) (SEQ ID NO: 89) and RCD186(5′-CTAGCTCGGTGTCCCGATGTCCACTGT-3′) (SEQ ID NO: 90). This PCR productwas cloned into pCR2.1 vector (Invitrogen, Carlsbad, Calif.) andsequenced. From this clone, a PCR overlap mutagenesis was performed forthree purposes: 1) to remove a NotI site within the open reading framewhile maintaining the wild-type protein sequence; 2) to truncate theprotein immediately downstream of the endogenous transmembrane domain;and 3) to introduce AscI and PacI sites at the 5′ and 3′ ends formodular cloning. To do this, the 5′ end of the gene up to the NotI sitewas amplified using primers RCD198(5′-CTTAGGCGCGCCGGCCGCGACCTGAGCCGCCTGCCC-3′) (SEQ ID NO: 91) and RCD201(5′-GGGGCATATCTGCCGCCCATC-3′) (SEQ ID NO: 92) and the 3′ end wasamplified with primers RCD200 (5′-GATGGGCGGCAGATATGCCCC-3′) (SEQ ID NO:93) and RCD199 (5′-CTTCTTAATTAACTAGCTCGGTGTCCCGATGTCCAC-3′) (SEQ ID NO:94). The products were overlapped together with primers 198 and 199 toresynthesize the ORF with the wild-type amino acid sequence whileeliminating the NotI site. The new truncated hGalTI PCR product wascloned into pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and sequenced.The introduced AscI/PacI sites were then used to subclone the fragmentinto plasmid pRCD259, which is a PpURA3/HYGR roll-in vector, to createpRCD260. A library of yeast targeting sequence transmembrane domains asdescribed herein, was ligated into the NotI/AscI sites located upstreamof the hGalTI gene to create plasmids pXB20-pXB67. pXB53 is a truncatedS. cerevisiae Mnn2(s) targeting peptide (1-108 nucleotides of MNN2 fromGenbank Accession No. NP_(—)009571) ligated in-frame to a 43 N-terminalamino acid deletion of a human β1,4-galactosyltransferase I (GenbankAccesion No. AH003575).

Using the above vectors, pJN711b (FIG. 29) was constructed, which is aHYGR plasmid containing hGalTI-53, POCH1-SpGALE, and DmUGT.

Generation of pSH326b for Expressing Human CMP-Sialic Acid Synthase(hCMP-Sia Syn) and Mouse CMP-Sialic Acid Transporter (mCMP-Sia Tr)

The gene encoding the human CMP-Sialic acid synthase (Genbank AccesionNo. AF397212) (referred to as hCMP-Sia syn) was amplified as a NotI-PacIfragment from human prostate cDNA. The conditions used for thermocyclingwere as follows: 95° C. 2 min, 1 cycle; 97° C. 30 sec, 60° C. 30 sec,72° C. 2.5 min, 25 cycles; 72° C. 5 min. using oligonucleotide primers:GGGAGAATGCGGCCGCCACCATGGACTCGGTGGAGAAGGGGGCCGCCAC CTC (hCMP-NANA synNotI/Koz) (SEQ ID NO: 95) andCCTTAATTAACTATTTTTGGCATGAATTATTAACTTTTTCCATTA (hCMP-NANA syn PacI) (SEQID NO: 96) with Advantage™ DNA polymerase (BD Biosciences). Theresulting 1.3 Kb fragment was cloned into pCR2.1 (Invitrogen), sequencedand designated pHW6. The hCMP-Sia syn was cloned from pHW6 into pPB140(a Kanamycin resistance vector, containing a 1.2 Kb fragment of P.pastoris HIS3 loci to facilitate ‘rolling-in’ integration, and theGAPDH-CYC promoter-terminator expression cassette) as a NotI-PacIfragment, giving the vector pSH301.

The gene encoding the mouse CMP-Sialic acid transporter (GenbankAccession No. Z71268) (referred to as mCMP-Sia Tr) was amplified asabove (using 2.5 min extension time) from mouse brain cDNA usingoligonucleotide primers: CGGAATTCCACCATGGCTCCGGCGAGAGAAAATGTCAG(mCMP-NANA trans Koz/for) (SEQ ID NO: 97) andCGGAATTCTCACACACCAATGATTCTCTCTTTTGAAG (mCMP-NANA trans rev) (SEQ ID NO:98). The resulting fragment was cloned into pCR2.1 (Invitrogen),sequenced and designated pSH194. The mCMP-Sia syn was digested frompSH194 with EcoRI and blunted with T4 DNA polymerase prior to subcloninginto pJN664, previously digested with NotI-PacI and blunted, giving thevector pSH306. This construct was used as template to amplify the PMAmCMP-Sia cassette flanked by XhoI sites using the primers:GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC (pPMA for XhoI) (SEQ ID NO: 99)and CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG (PMAtt rev XhoI) (SEQ ID NO:100). The resulting vector was designated pSH317.

The 2.6 Kb XhoI fragment from pSH317, containing the PMA mCMP-Siatransporter cassette, was ligated into the XhoI site of pSH301(containing hCMP-Sia syn) to give the double expression cassette vectorpSH326b (FIG. 30).

Generation of pSH370 for Expressing Rat ST6Gal

An N-terminal deletion of the rat ST6Gal (Genbank Accession No. M18769)was amplified from rat liver cDNA using the conditions described abovefor the hGNE, but using a 2 min extension time instead of 5 min. Theprimers were: GGCGCGCCAGCAAGCAAGACCCTAAGGAAGACATTCC (rST6GalI d63 AscI)(SEQ ID NO: 101) and CCTTAATTAATCAACAACGAATGTTCCGGAAGCCAGAAAGG (rST6GalIPacI) (SEQ ID NO: 102). The resulting fragment was cloned into pCR2.1(Invitrogen), sequenced and designated pSH271. Subsequently, the 1 kbfragment containing the encoded rST6Gal catalytic domain was subclonedinto a nourseothricin selectable vector (Hansen, 2003) generating afusion of the rST6Gal to the first 108 base pairs of Mnn2 (leader 53),under the control of the PMA promoter. The resulting vector wasdesignated pSH370 (FIG. 31).

Generation of pSH373 for Expressing Mannosidase II (MannII) and GnTII

D. melanogaster mannosidase II (Genbank Accession No. X77652) fused toleader 53 (KD53) was subcloned as a NotI-PacI fragment from pKD53 intothe expression vector pJN702. The resulting vector was designatedpSH368.

The rat GnTII (Genbank Accession No. U21662) fused to the first 108 basepairs of Mnn2 (leader 53) was digested from pTC53 (see Hamilton, 2003)as a NotI-PacI fragment and subcloned into pJN664 digested with the sameenzymes. The resulting vector was designated pSH327.

The 2.7 kb SwaI fragment of pSH327, containing the PMA GnTII expressioncassette, was ligated into the PmeI site of pSH368. The resulting vectorwas designated pSH373 (FIG. 32).

Generation of pSH568 for Expressing Codon-Optimized Human ST6Gal

A codon-optimized version of an N-terminal deletion of human ST6Gal(Genbank Accession No. NM_(—)003032) fused to the first 108 base pairsof Mnn2 (leader 53) was generated by GENEART GmbH (Regensburg Germany).The nucleic acid and amino acid sequences of this codon optimizedhST6Gal are shown in FIG. 33. The resulting NotI-PacI fragment wassubcloned into the expression vector pJN703b, giving the constructpSH568 (FIG. 34).

pSH568 was digested with SfiI and transformed into the strain YSH272,which was generated in a similar fashion to YSH160, except that theintroduction of rST6Gal was omitted and the vector pSH505 was usedinstead of pSH373. In vector pSH505, the MannII and GntII genes wereintroduced as leader 4 and leader 5 fusions, respectively. Leader 4comprises S. cerevisiae mns1s leader, and leader 5 comprises S.cerevisia mns1m, respectively. See US 2002/0137134.

Engineering In Vivo Transfer of Sialic Acid to N-Glycans in Yeast

P. pastoris YSH1 (Hamilton, 2003) was used as the initial host strain toengineer the glycosylation machinery necessary to produce sialylatedglycoproteins. YSH1 (Ura3-) was transformed with pSH321 (hGNE, hSiaPsyn,URA3; described above) digested with ApaI, and a positive clonedesignated YSH103 was identified using Ura3 as the selectable marker.YSH103 was then transformed with SfiI digested pJN711b (hGalTI-53,POCH1-SpGALE, DmUGT, Δhis1::Hyg; described above), using Hyg as theselectable marker. A transformant designated YSH116 (Δhis1) was selectedand transformed with BspEI digested pSH326 (hCMP-Sia syn, mCMP-Sia Tr;described above), using Kan as the selectable marker. A transformantdesignated YSH125 (Δhis1) was selected and transformed with pSH370(rST6Gal; described above) digested with EcoRI and a positive cloneselected, using Nat as a selectable marker (Hansen, 2003). Atransformant designated as YSH145 (Δhis1) was selected and subsequentlytransformed with SfiI digested pSH373 (MannII, GnTII, (Δarg1::His1);described above), using this as a selectable marker. The resultingtransformant designated as YSH160 (Δarg1) produced glycoproteinsexhibiting the oligosaccharide structure: NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ asshown in FIG. 36.

Alternatively, YSH125 (Δhis1) was selected and transformed with pSH568(codon optimized hST6Gal) digested with SfiI and a positive cloneselected using Nat as a selectable marker. A transformant was selectedand subsequently transformed with SfiI digested pSH505 (MannII, GnTII,(Δarg1::His1); described above). The resulting transformant designatedas YSH272 (Δarg1) produced glycoproteins exhibiting the oligosaccharidestructure: NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ as shown in FIG. 37.

All of the yeast strains were transformed by electroporation (asrecommended by the manufacturer of the electroporator BioRad).Integration into the host genome was confirmed by PCR (Nett, 2005).

Glycan Analysis

Oligosaccharides on a recombinant Kringle 3 glycoprotein expressed ineach of the strains described above were analyzed using MALDI-TOF MS.

Glycan analysis of glycoproteins produced in strain YSH126, in whichsrain YSH116 is further transformed with pSH326, showed mass consistentwith the glycan structure GalGlcNAcMan₅GlcNAc₂ confirming the in vivotransfer of a terminal galactose residue (FIG. 35).

Glycan analysis of glycoproteins produced in strain YSH145 showed massconsistent with the glycan structure NANAGalGlcNAcMan₅GlcNAc₂ confirmingthe in vivo transfer of sialic acid onto at least one oligosaccharidebranch (FIGS. 35 and 36).

Glycan analysis of glycoproteins produced in strain YSH160 showed massconsistent with the glycan structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂confirming the in vivo transfer of sialic acid onto at least oneoligosaccharide branch (FIG. 36).

Glycan analysis of glycoproteins produced in strain YSH272 showed massconsistent with the glycan structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂confirming the in vivo transfer of sialic acid onto at least oneoligosaccharide branch (FIG. 37).

Table 13 summarizes the extent of in vivo sialic acid transfer onto arecombinant glycoprotein in an engineered P. pastoris strain madeaccording to the invention disclosed herein. Values represent thepercentages of total glycans that contained sialic acid; and thussensitive to sialidase treatment.

TABLE 13 In Vivo Sialic Acid Transfer In Yeast Strain Mono-sialylatedBi-sialylated Total transfer YSH145 15% — 15% YSH160 20%  2% 22% YSH27234% 24% 58%

Materials and Method for the Experiments Described in Example 17

MOPS, sodium cacodylate, manganese chloride, UDP-galactose andCMP-N-acetylneuraminic acid were from Sigma. TFA was from Aldrich.β1,4-galactosyltransferase from bovine milk were from Calbiochem.Protein N-glycosidase F, mannosidases, and oligosaccharides were fromGlyko (San Rafael, Calif.). DEAE ToyoPearl resin was from TosoHaas.Metal chelating “HisBind” resin was from Novagen (Madison, Wis.).96-well lysate-clearing plates were from Promega (Madison, Wis.).Protein-binding 96-well plates were from Millipore (Bedford, Mass.).Salts and buffering agents were from Sigma (St. Louis, Mo.). MALDImatrices were from Aldrich (Milwaukee, Wis.).

Shake-Flask Cultivations

A single colony was picked from an YPD plate (<2 weeks old) containingthe strain of interest and inoculated into 10 ml of BMGY media in a 50ml “Falcon” centrifuge tube. The culture was grown to saturation at 24°C. (approx. 48 hours). The seed culture is transferred into a 500 mlbaffled volumetric flask containing 150 ml of BMGY media and grown toOD600 of 5±2 at 24° C. (approx. 18 hours). The growth rate of the cellswas determined as the slope of a plot of the natural logarithm of OD600against time. The cells were harvested from the growth medium (BMGY) bycentrifugation at 3000 g for 10 minutes, washed with BMMY and suspendedin 15 ml of BMMY in a 250 ml baffled volumetric flask. After 24 hours,the expression medium flask is harvested by centrifugation (3000 g for10 minutes) and the supernatant analyzed for K3 production.

Bioreactor Cultivations

A 500 ml baffled volumetric flask with 150 ml of BMGY media wasinoculated with 1 ml of seed culture (see flask cultivations). Theinoculum was grown to an OD600 of 4-6 at 24° C. (approx 18 hours). Thecells from the inoculum culture was then centrifuged and resuspendedinto 50 ml of fermentation media (per litre of media: CaSO4.2H2O 0.30 g,K2SO4 6.00 g, MgSO4.7H2O 5.00 g, Glycerol 40.0 g, PTM1 salts 2.0 ml,Biotin 4×10-3 g, H3PO4 (85%) 30 ml, PTM1 salts per litre: CuSO4.H2O6.00, NaI 0.08 g, MnSO4.7H2O 3.00 g, NaMoO4.2H2O 0.20 g, H3BO3 0.02 g,CoCl2.6H2O 0.50 g, ZnCl2 20.0 g, FeSO4.7H2O 65.0 g, Biotin 0.20 g, H2SO4(98%) 5.00 ml).

Fermentations were conducted in 3 liter dished bottom (1.5 liter initialcharge volume) Applikon bioreactors. The fermentors were run in afed-batch mode at a temperature of 24° C., and the pH was controlled at4.5±0.1 using 30% ammonium hydroxide. The dissolved oxygen wasmaintained above 40% relative to saturation with air at 1 atm byadjusting agitation rate (450-900 rpm) and pure oxygen supply. The airflow rate was maintained at 1 vvm. When the initial glycerol (40 g/l) inthe batch phase is depleted, which is indicated by an increase of DO, a50% glycerol solution containing 12 ml/l of PTM1 salts was fed at a feedrate of 12 ml/l/h until the desired biomass concentration was reached.After a half an hour starvation phase, the methanol feed (100% Methanolwith 12 ml/l PTM1) is initiated. The methanol feed rate is used tocontrol the methanol concentration in the fermentor between 0.2 and0.5%. The methanol concentration is measured online using a TGS gassensor (TGS822 from Figaro Engineering Inc.) located in the offgass fromthe fermentor. The fermentors were sampled every eight hours andanalyzed for biomass (OD600, wet cell weight and cell counts), residualcarbon source level (glycerol and methanol by HPLC using Aminex 87H) andextracellular protein content (by SDS page, and Bio-Rad protein assay).

Reporter Protein Expression, Purification and Release of N-LinkedGlycans

The K3 domain, under the control of the alcohol oxidase 1 (AOX1)promoter, was used as a model protein and was purified using the6×Histidine tag as reported previously (Choi, 2003). The glycans werereleased and separated from the glycoproteins by a modification of apreviously reported method (Papac and Briggs 1998). After the proteinswere reduced and carboxymethylated, and the membranes blocked, the wellswere washed three time with water. The protein was deglycosylated by theaddition of 30 μl of 10 mM NH4HCO3 pH 8.3 containing one milliunit ofN-glycanase (Glyko). After 16 hr at 37° C., the solution containing theglycans was removed by centrifugation and evaporated to dryness.

Protein Purification

Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle3 was purified from expression media using a C-terminal hexa-histidinetag. The robotic purification is an adaptation of the protocol providedby Novagen for their HisBind resin. Briefly, a 150 uL (μL) settledvolume of resin is poured into the wells of a 96-well lysate-bindingplate, washed with 3 volumes of water and charged with 5 volumes of 50mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole,0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media isdiluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) andloaded onto the columns. After draining, the columns are washed with 10volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole,0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HClpH7.9). The eluted glycoproteins are evaporated to dryness bylyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by amodification of a previously reported method (Papac, et al. A. J. S.(1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP(Immobilon-P membrane) plate (Millipore) are wetted with 100 uL ofmethanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8Murea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum aftereach addition. The dried protein samples are dissolved in 30 uL of RCMbuffer and transferred to the wells containing 10 uL of RCM buffer. Thewells are drained and washed twice with RCM buffer. The proteins arereduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37°C. The wells are washed three times with 300 uL of water andcarboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30min in the dark at room temperature. The wells are again washed threetimes with water and the membranes blocked by the addition of 100 uL of1% PVP 360 in water for 1 hr at room temperature. The wells are drainedand washed three times with 300 uL of water and deglycosylated by theaddition of 30 uL of 10 mM NH4HCO3 pH 8.3 containing one milliunit ofN-glycanase (Glyko). After 16 hours at 37° C., the solution containingthe glycans was removed by centrifugation and evaporated to dryness.

Miscellaneous

Proteins were separated by SDS/PAGE according to Laemmli (Laemmli 1970).

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

Molecular weights of the glycans were determined using a Voyager DE PROlinear MALDI-TOF (Applied Biosciences) mass spectrometer using delayedextraction. The dried glycans from each well were dissolved in 15 uL ofwater and 0.5 uL spotted on stainless steel sample plates and mixed with0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowedto dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm)with a 4 ns pulse time. The instrument was operated in the delayedextraction mode with a 125 ns delay and an accelerating voltage of 20kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, theinternal pressure was less than 5×10-7 torr, and the low mass gate was875 Da. Spectra were generated from the sum of 100-200 laser pulses andacquired with a 2 GHz digitizer. Man5GlcNAc2 oligosaccharide was used asan external molecular weight standard. All spectra were generated withthe instrument in the positive ion mode. The estimated mass accuracy ofthe spectra was 0.5%.

Example 18 Engineering K. lactis Cells To Produce N-Glycans with theStructure Man₅GlcNAc₂

Identification and Disruption of the K. lactis OCH1 Gene

The OCH1 gene of the budding yeast S. cerevisiae encodes a1,6-mannosyltransferase that is responsible for the first Golgilocalized mannose addition to the Man₈GlcNAc₂ N-glycan structure onsecreted proteins (Nakanishi-Shindo, 1993). This mannose transfer isgenerally recognized as the key initial step in the fungal specificpolymannosylation of N-glycan structures (Nakanishi-Shindo et al., 1993;Nakayama, 1992; Morin-Ganet, 2000). Deletion of this gene in S.cerevisiae results in a significantly shorter N-glycan structure thatdoes not include this typical polymannosylation or a growth defect atelevated temperatures (Nakayama, 1992).

The Och1p sequence from S. cerevisiae was aligned with known homologsfrom Candida albicans (Genbank Accession No. AAL49987), and P. pastoris(Choi, 2003) along with the Hoc1 proteins of S. cerevisiae (Neiman,1997) and K. lactis (PENDANT EST database) which are related butdistinct mannosyltransferases. Regions of high homology that were incommon among Och1p homologs but distinct from the Hoc1p homologs wereused to design pairs of degenerate primers that were directed againstgenomic DNA from the K. lactis strain MG1/2 (Bianchi, 1987). PCRamplification with primers RCD33 (CCAGAAGAATTCAATTYTGYCARTGG) (SEQ IDNO:34) and RCD34 (CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO:35) resulted ina 302 bp product that was cloned and sequenced and the predictedtranslation was shown to have a high degree of homology to Och1 proteins(>55% to S. cerevisiae Och1p).

The 302 bp PCR product was used to probe a Southern blot of genomic DNAfrom K. lactis strain (MG1/2) with high stringency (Sambrook et al.,1989). Hybridization was observed in a pattern consistent with a singlegene indicating that this 302 bp segment corresponds to a portion of theK. lactis genome and K. lactis (KlOCH1) contains a single copy of thegene. To clone the entire KlOCH1 gene, the Southern blot was used to mapthe genomic locus. Accordingly, a 5.2 kb BamHI/PstI fragment was clonedby digesting genomic DNA and ligating those fragments in the range of5.2 kb into pUC19 (New England Biolabs, Beverly, Mass.) to create a K.lactis subgenomic library. This subgenomic library was transformed intoE. coli and several hundred clones were tested by colony PCR using RCD33/34. The 5.2 kb clone containing the predicted KlOCH1 gene wassequenced and an open reading frame of 1362 bp encoding a predictedprotein that is 46.5% identical to the S. cerevisiae OCH1 gene. The 5.2kb sequence was used to make primers for construction of anoch1::KAN^(R) deletion allele using a PCR overlap method (Davidson,2002). This deletion allele was transformed into two K. lactis strainsand G418 resistant colonies selected. These colonies were screened byboth PCR and for temperature sensitivity to obtain a strain deleted forthe OCH1 ORF. The results of the experiment show strains which reveal amutant PCR pattern, which were characterized by analysis of growth atvarious temperatures and N-glycan carbohydrate analysis of secreted andcell wall proteins following PNGase digestion. The och1 mutationconferred a temperature sensitivity which allowed strains to grow at 30°C. but not at 35° C. FIG. 12A shows a MALDI-TOF analysis of a wild typeK. lactis strain producing N-glycans of Man₈GlcNAc₂ [c] and higher.

Identification, Cloning, and Disruption of the K. lactis MNN1 Gene

S. cerevisiae MNN1 is the structural gene for the Golgiα-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid typeII membrane protein (Yip, 1994). Both N-linked and O-linkedoligosaccharides isolated from mnn1 mutants lack α-1,3-mannose linkages(Raschke, 1973).

The Mnn1p sequence from S. cerevisiae was used to search the K. lactistranslated genomic sequences (PEDANT). One 405 bp DNA sequence encodinga putative protein fragment of significant similarity to Mnn1p wasidentified. An internal segment of this sequence was subsequently PCRamplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ ID NO:36)and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO:37) and used to probea Southern blot of genomic DNA from K. lactis strain (MG1/2). Based onthe Southern hybridization data a 4.2 Kb BamHI-PstI fragment was clonedby generating a size-selected library as described herein. A singleclone containing the K. lactis MNN1 gene was identified by whole colonyPCR using primers KMN1 (SEQ ID NO:36) and KMN2 (SEQ ID NO:37) andsequenced. Within this clone a 2241 bp ORF was identified encoding apredicted protein that was 34% identical to the S. cerevisiae MNN1 gene.Primers were designed for construction of a mnn1::NAT^(R) deletionallele using the PCR overlap method (Davidson, 2002).

This disruption allele was transformed into a strain of K. lactis byelectroporation and Noursethoicin resistant transformants were selectedand PCR amplified for homologous insertion of the disruption allele.Strains that reveal a mutant PCR pattern may be subjected to N-glycancarbohydrate analysis of a known reporter gene.

FIG. 12B depicts the N-glycans from the K. lactis och1 mnn1 deletionstrain observed following PNGase digestion and MALDI-TOF as describedherein. The predominant peak at 1908 (m/z) indicated as [d] isconsistent with the mass of Man₉GlcNAc₂.

Example 19 Engineering Plant Cells to Express GlcNAc Transferases orGalactosyltransferases

GlcNAc transferase IV is required for the addition of β1,4 GlcNAc to theα-1,6 mannose residue and the α-1,3 mannose residues in complexN-glycans in humans. So far, GlcNAc transferase IV has not been detectedin or isolated from plants. A transgenic plant that is capable of addinghuman-like N-glycans must therefore be engineered to express GlcNActransferase IV. Thus, the plant host cell or transgenic plant must alsolocalize an expressed GlcNAc transferase IV to the correct intracellularcompartment in the host so that the enzyme can add the β1,4 GlcNAc tothe appropriate mannose residues.

There is some evidence that glycosyltransferases from mammals and plantshave similar targeting signals. For example, a full-length ratα-2,6-sialyltransferase has been shown to correctly localize to thetrans Golgi network in transgenic Arabidopsis plant cells, but was notnecessarily active there (Wee, 1998). A fusion construct havingfifty-two N-terminal amino acids from α-2,6-sialyltransferase fused to agreen fluorescent reporter protein (GFP) was also shown to correctlylocalize to the plant Golgi (Boevink, 1998). Two mammalianproteins—TGN30 and furin—and AtELP, an Arabidopsis integral membraneprotein (Sanderfoot, 1998), which localize to the trans Golgi network,each contain a tyrosine tetrapeptide motif which targets them to theGolgi, probably by a recycling mechanism via the plasma membrane.Although mammals and plants appear to share some common mechanismsrelated to protein targeting, exogenous glycosylases may nonetheless nottarget correctly in a plant cell, however, localization does notnecessarily equate with enzyme activity. It therefore becomes essentialto devise means to correctly target in a plant cell these enzymes and/orother enzymes that participate in forming complex, human-like N-glycans.

Glycosylation enzymes are integral membrane proteins which reside in theendoplasmic reticulum and Golgi apparatus. The targeting andlocalization signals are normally contained in the cytoplasmic and/ortransmembrane domains and in some cases are contained in some lumenaldomains. For example, fifty-two amino acids that make up thetransmembrane domain, nine cytoplasmic amino acids and twenty-sixlumenal amino acids of α-2,6-sialyltransferase are required to targetGFP to the trans Golgi network (Boevink, 1998).

Thus, a library of sequences encoding cellular targeting signal peptidescomprising either just the cytoplasmic and transmembrane domains or thecytoplasmic, transmembrane and lumenal domains of endoplasmic reticulumand Golgi specific proteins is generated, as described in Example 11.The targeting peptide sequences may be chosen from ER and Golgi-residentplant, yeast or animal proteins. A glycosylation related protein, e.g.,an enzyme (or catalytic domain thereof) such as a glycosylase orintegral membrane enzyme can be fused in-frame to the library oftargeting peptide sequences and introduced into plants (FIG. 13). Planttargeting peptide sequences may be most efficient in localizing thechimeric enzymes to the ER and Golgi, although targeting peptidesequences from fungi and mammals may also be effective. For example, theN-terminal 77 amino acids from tobacco N-acetylglucosaminyl TransferaseI have been shown to correctly target a reporter protein to the Golgi(Essl, 1999). In one embodiment, one or more N-terminal fragmentscomprising these 77 amino acids (or subsets of these amino acids) isfused to one or more fragments comprising a catalytic domain of GlcNActransferase IV. At least one resulting fusion protein correctlylocalizes a functional GlcNAc transferase IV to the Golgi apparatus in aplant cell, as evidenced by monitoring the glycosylation state of areporter glycoprotein resident or introduced into the plant host cellusing techniques described herein.

Another plant enzyme shown to localize to the Golgi is ArabidopsisGlcNAc transferase II (Strasser, 1999). Thus, in another embodiment, oneor more different fragments of the Arabidopsis GlcNAc transferase IItargeting peptide are fused to a GlcNAc transferase IV catalytic domainand fusion constructs produced and tested as described above. The plantspecific β1,2-xylosyltransferase from Arabidopsis thaliana is anotherprotein that localizes to the Golgi and its localization and retentionin the Golgi is dependent on its cytoplasmic and transmembrane sequences(Dirnberger, 2002). Thus, in another embodiment, one or more fragmentscomprising the cytoplasmic and transmembrane sequences ofβ1,2-xylosyltransferase are fused to one or more fragments comprising aGlcNAc transferase IV catalytic domain and resulting fusion constructsare transformed into plant cells and tested for their ability to producea human-like N-glycan and to otherwise modulate glycosylation in theplant host cell.

Because GlcNAc transferase IV or Galactosyltransferase from one organismmay function more efficiently in a specific plant host than one fromanother organism, fragments comprising GlcNAc transferase IVs (orcatalytic domains) from various eukaryotic organisms are fused in-frameto the library of endoplasmic reticulum (ER) and Golgi targeting peptidesequences and are then introduced into plants. The use of a library ofnucleic acids encoding enzyme domains isolated or derived from differentspecies increases the chances of efficient glycosylation—in addition tocorrect localization and glycosylation by GlcNAc transferase IV.

The methods and combinatorial nucleic acid libraries of the inventionmay be used to introduce and localize, sequentially or en masse,multiple enzymes required to glycosylate proteins in a plant cell withhuman-like N-glycans. As different plant species may require differentgrowth conditions, protocols for transformation may vary depending onthe species being transformed (Potrykus, 1990). The commonly usedmethods for generating transgenic plants include Agrobacterium mediatedtransformation, particle bombardment (Sanford, 1990) andelectroporation.

Agrobacterium Method

The catalytic domains of GlcNAc transferase IVs are fused in-frame tomultiple different targeting peptide sequences known to target proteinsto the ER and Golgi in plants. Each of these fusion constructs isintroduced under the control of the ubiquitously expressed promoterslike the 35S CaMV, ubiquitin or actin promoters, tissue specificpromoters or inducible promoters. A plant specific terminator region isalso used. This cassette (promoter::targeting peptide-GlcNAc transferaseIV::terminator) is cloned into a vector suitable for Agrobacteriummediated transformation (FIG. 13). The vector also contains a selectablemarker that allows one to select for transformed plants. The commonselectable markers used include those resulting in kanamycin, hygromycinand basta resistance. The construct is introduced into Agrobacterium viawell-established transformation methods, which are available in the art.An Agrobacterium library of Golgi-targeted GlcNAc transferase IVs isthereby generated.

Embryonic and meristematic tissue may be transformed and can regeneratetransgenic plants. To transform tissue, tissue explants (these could beplumules and radicals from germinated seeds) are first soaked and coatedwith an Agrobacterium innoculum. They are then cultured on platescontaining the innoculum to form an undifferentiated mass of cellstermed the callus. Transformed plant cells are selected for by adding tothe medium the relevant kanamycin, hygromycin or basta (depending on theselectable marker used on the construct). The transformed plant cellscan either be grown in culture and remain undifferentiated or they aretreated with shoot regenerating and shoot elongation medium. Explantsthat differentiate are transferred onto rooting medium to generatetransgenic plants. Some plants like Arabidopsis can be transformed bydipping flowers into an Agrobacterium solution. Seeds from thetransformed plants are germinated on plates containing the relevantherbicide or antibiotic selection. Transgenic plants are those that growon the selection media. The transgenic plants are then screened forthose with properly glycosylated proteins (i.e., those which havecomplex, human-like N-glycans) by isolating glycoproteins from plantextracts and analyzing glycoprotein patterns as described elsewhereherein, e.g., by using a specific antibody or lectin. Although theAgrobacterium method is economical and simple, it is limited to certainspecies of plants. Accordingly, plants that cannot be transformed usingAgrobacterium can be transformed by ballistics or electroporation.

Particle Bombardment Method and Electroporation

Compared to Agrobacterium mediated transformation, these methods have agreater tendency to insert multiple copies of the transgene into thegenome. This could result in gene silencing and cosuppression. However,unlike Agrobacterium mediated transformation, these methods are notspecies limited and are therefore useful when an Agrobacterium methodcannot be employed to generate transgenic plants. In the particlebombardment method, cultured plant cells are bombarded with very smalltungsten or gold particle that have been coated with DNA(promoter::targeting peptide-GlcNAc transferase IV-terminator::selectable marker) (FIG. 13) (rb and lb not required) while in theelectroporation method, plant cells in a DNA (promoter::targetingpeptide-GlcNAc transferase IV-terminator::selectable marker) solutionare treated with an electric pulse that perforates the cell, allowing itto take up DNA. The cells are then cultured and allowed to recover.Stable transformants are selected for by culturing and regeneratingplants on appropriate selection medium.

Engineering Soybeans to Express GlcNAc Transferase IV Using a SoybeanCotyledonary Node Agrobacterium Mediated Transformation System

An Agrobacterium library of Golgi-targeted GlcNAc transferase IV isgenerated as described above. Soybean explants are transformed with thelibrary using a protocol described by Hinchee, 1988. A reporter proteinis expressed with a His tag, purified and then analyzed. Transgenicplants are assayed for proteins with the α-1,6 mannose and the α-1,3mannose residues using, e.g., mass spectroscopy.

Engineering Pea to Express GlcNAc Transferase IV Using ParticleBombardment

A GlcNAc transferase IV plasmid library is coated onto tungsten or goldparticles and used as microprojectiles to bombard calli derived from peaembryonic tissue as described (Molnar, 1999). A reporter protein isexpressed with a His tag, purified and then analyzed. Transgenic plantsare assayed for proteins with the α-1,6 mannose and the α-1,3 mannoseresidues using, e.g., MALDI.

Engineering Plants to Express GlcNAc Transferase I

GlcNAc transferase I is involved in the addition of GlcNAc to theterminal α-1,3 mannose residue to form Man₅GlcNAc₂, an essential step inthe maturation of complex N-glycans. Although GlcNAc transferase I hasbeen isolated from plants and appears to have the same function as itsmammalian homolog, it may not be the most efficient enzyme forglycosylation of mammalian or exogenous proteins and may not be found inevery plant species. As the addition of GlcNAc to the terminal α-1,3mannose residue is a controlling step in the mammalian glycosylationpathway, it is advantageous to have transgenic plants that can carry outthis step efficiently. To create transgenic plants that express GlcNActransferase I that can function efficiently to promote the formation ofcomplex N-glycans, a library of GlcNAc transferase I isolated or derivedfrom various organisms is fused in-frame to multiple plant Golgitargeting peptide sequences according to the methods described herein.The combinatorial library thus created is introduced into a plant cellor organism as described above for GlcNAc transferase IV.

Engineering Maize to Express GlcNAc Transferase I Using ParticleBombardment

Transgenic maize can be obtained using a protocol similar to the oneused to generate peas that express GlcNAc transferase IV. Here theGlcNAc transferase I plasmid library is coated onto tungsten or goldparticles and used to bombard calli derived from maize embryonic tissue,e.g., using a protocol specific for the generation of transgenic maize(Gordon-Kamm, 1990). Transgenic plants are assayed for proteins havingGlcNAc on the terminal α-1,3 mannose residue, e.g., using specificantibodies or by assaying reduced binding of the N-glycans to certainlectins or by using MALDI-TOF.

Other useful references for using plant host cells according to theinvention include: Dirnberger, 2002; Frame, 2002; Gomord, 1999; Laursen,1994; Orci, 2000; Newell, 2002; Pawlowski, 1996; Schroeder, 1993;Sorokin, 2000; Strasser, 1999; Tomes, 1990.

Engineering Plant Cells to Produce β1,4-Galactosyltransferases

β1,4-galactosyltransferase is an important human glycosyltransferasethat is absent in plants. Lerouge, 1998. In mammals,β1,4-galactosyltransferase is localized in the Golgi and is responsiblefor the transfer of galactose residues to the terminalN-acetylglucosamine of the core Man₃GlcNAc₂ of complex N-glycans. Inplants, the Man₃GlcNAc₂ core contains β1,2-xylose and α1,3-fucoseresidues and lacks the β1,4-galactose. The xylose and fucosemodifications are implicated in allergies and act as antigenic epitopesand are therefore not desirable modifications of therapeutic proteins.

The galactose modifications carried out by β1,4-galactosyltransferasecan be important for the proper functioning of the therapeutic proteins.In mammals, β1,4-galactosyltransferase acts afterN-acetylglucosaminyltransferase I and N-acetylglucosaminyltransferase IIand has been shown to initiate branching of the complex N-glycan.Lerouge, 1998; Palacpac, 1999. In tobacco cells, expression of humanβ1,4-galactosyltransferase has been shown to result in galactosylatedN-glycans with reduced fucose and xylose modifications. Bakker, 2001;Fujiyama, 2001; Palacpac, 1999. In these studies, a 1.2 kb fragment ofhuman β1,4-galactosyltransferase was cloned downstream of thecauliflower mosaic virus promoter (35SCaMV), introduced into the binaryvector pGA482, and finally into tobacco cells. Palacpac, 1999.

Tobacco cells were transformed using the agrobacterium method describedby Rempel et al. (Rempel, 1995). Transformation of tobacco cells hasalso been described (An, 1985). Expression of β1,4-galactosyltransferaseunder the 35SCaMV resulted in ubiquitous expression of the gene intobacco cells. Tobacco cells expressing human β1,4-galactosyltransferaseshowed the presence of galactosylated N-glycans. (Palacpac, 1999; Bakker1991), showed that crossing tobacco plants expressing humanβ1,4-galactosyltransferase with plants expressing the heavy and lightchain of a mouse antibody resulted in plants in which the antibodyshowed 30% galactosylation (Bakker, 2001).

A combinatorial DNA library can be constructed to obtain aβ1,4-galactosyltransferase plant cell line for the addition of galactoseresidues. The combinatorial DNA library can effectively produce celllines which are more efficient in the addition of galactose residues.Once such a cell line is made it can be easily crossed to cell linesexpressing other glycosylation enzymes and to those expressingtherapeutic proteins to produce therapeutic proteins with human-likeglycosylation. The final line can then be grown as plants and harvestedto extract proteins or can be cultured as plant cells in suspensioncultures to produce proteins in bioreactors. By expressing thetherapeutic proteins using the library of signal peptides, it ispossible to retain the therapeutic protein within the cells or have themsecreted into the medium. Tobacco cells expressingβ1,4-galactosyltransferase secrete galactosylated N-glycans (Ryo, 2002).While horseradish peroxidase isozyme C expressed in tobacco plantsexpressing β1,4-galactosyltransferase contained xylose and fucosemodifications, no xylose or fucose modification could be detected inhorseradish peroxidase isozyme C expressed in tobacco cells expressingβ1,4-galactosyltransferase (GT6 cells). (Fujiyama, 2001). This indicatesthat it may be advantageous to express therapeutic proteins in celllines instead of whole plants.

Engineering Plants to Produce Sialyltransferase

In mammals, sialyltransferase is a trans Golgi enzyme that adds terminalsialic acid residues to glycosylated polypeptides. Thus far, terminalsialic acid residues have not been detected in plants (Wee, 1998). Weeet al. expressed the rat α-2,6-sialyltransferase in transgenicArabidopsis and showed that the enzyme properly localized to the Golgiand was functional. Wee et al. demonstrated that membranes isolated fromtransgenic Arabidopsis, when incubated with CMP-³H-sialic acid andasialofetuin acceptor, resulted in the addition of sialic acid residueswhile membrane isolated from wild-type Arabidopsis did not. Whileexpressing the rat α-2,6-sialyltransferase in Arabidopsis resulted in afunctional enzyme that was able to incorporate sialic acid residues,fusing the mammalian enzymes α-2,3-sialyltransferase andα-2,6-sialyltransferase to a variety of transit peptides using thelibrary approach of the present invention (described above) can resultin more efficient sialylation in other plant species. Wee et al. had toisolate membranes and incubate them with CMP-³H-sialic acid andasialofetuin acceptor since Arabidopsis does not have CMP-sialic acid orits transporter. In order to overcome this additional step and obtainsialic acid addition in the plant, CMP-sialic acid biosynthetic pathwayand the CMP-sialic acid transporter can be co-expressed in transgenicplants expressing α-2,3-sialyltransferase and α-2,6-sialyltransferase(see Examples 6, 16 and 17). As an alternative, the CMP-sialic acidtransporter can be co-expressed with α-2,3-sialyltransferase andα-2,6-sialyltransferase in plant cells grown in suspension culture, andCMP-sialic acid or other precursors of CMP-sialic acid supplied in themedium.

Expressing α-2,3-Sialyltransferase and α-2,6-Sialyltransferase in Lemna

As described in the U.S. Pat. No. 6,040,498 (“the '498 patent”), lemna(duckweed) can be transformed using both agrobacterium and ballisticmethods. Using protocols described in the '498 patent, lemna will betransformed with a library of Golgi targeted α-2,3-sialyltransferaseand/or α-2,6-sialyltransferase and a library of mammalian CMP-sialicacid transporters. Transgenic plants can be assayed for those thatproduce proteins with terminal sialic acid residues according toscreening techniques discussed herein (Example 17).

Expressing α-2,3-Sialyltransferase and α-2,6-Sialyltransferase inTobacco Cells

Alpha-2,3-sialyltransferase and/or α-2,6-sialyltransferase and/or alibrary of mammalian CMP-sialic acid transporters can also be introducedinto tobacco cells grown in suspension culture as described forβ1,4-galactosyltransferases. CMP-sialic acid can be added to the medium.Both the cells and the culture medium (secreted proteins) can be assayedfor proteins with terminal sialic acid residues according to screeningtechniques discussed herein (Example 17).

Example 20 Engineering Insect Cells to Produce Glycosyltransferases

Insect cells provide another mechanism for producing glycoproteins butthe resulting glycoproteins are not complex human-like glycoproteins.(Marz, 1995; Jarvis, 1997.) It is another feature of the presentinvention to provide enzymes in insect cells, which are targeted to theorganelles in the secretory pathway. In a preferred embodiment, enzymessuch as glycosyltransferases, galactosyltransferases andsialyltransferases are targeted to the ER, Golgi or the trans Golginetwork in lepidopteran insect cells (Sf9). Expression of mammalianβ1,4-galactosyltransferase has been shown in Sf9 cells. Hollister, 1998.These enzymes are targeted by means of a chimeric protein comprising acellular targeting signal peptide not normally associated with theenzyme. The chimeric proteins are made by constructing a nucleic acidlibrary comprising targeting sequences as described herein and theglycosylation enzymes. Baculovirus expression in insect cells iscommonly used for stable transformation for adding mammalianglycosyltransferases in insect cells. (Hollister, 2001.)

TABLE 11 DNA And Protein Sequence Resources  1. European BioinformaticsInstitute (EBI) (a centre for research and services in bioinformatics) 2. Swissprot database  3. List of known glycosyltransferases and theirorigin.  4. human cDNA, Kumar et al (1990) Proc. Natl. Acad. Sci. U.S.A.87:9948-9952  5. human gene, Hull et al (1991) Biochem. Biophys. Res.Commun. 176:608-615  6. mouse cDNA, Kumar et al (1992) Glycobiology2:383-393  7. mouse gene, Pownall et al (1992) Genomics 12:699-704  8.murine gene (5′ flanking, non-coding), Yang et al (1994) Glycobiology5:703-712  9. rabbit cDNA, Sarkar et al (1991) Proc. Natl. Acad. Sci.U.S.A. 88:234-238 10. rat cDNA, Fukada et al (1994)Biosci.Biotechnol.Biochem. 58:200-201 1,2 (GnTII) EC 2.4.1.143 11. humangene, Tan et al (1995) Eur. J. Biochem. 231:317-328 12. rat cDNA,D'Agostaro et al (1995) J. Biol. Chem. 270:15211-15221 13. arabadopsiscDNA Strasser et al (1999) J. Glycoconj. 16 :787-791 14. C. elegans geneChen et al., (2002) Biochim. Biophys. Acta. 1573:271-279 β1,4 (GnTIII)EC 2.4.1.144 15. human cDNA, Ihara et al (1993) J. Biochem.113:692-69816. murine gene, Bhaumik et al (1995) Gene 164:295-300 17. rat cDNA,Nishikawa et al (1992) J. Biol. Chem. 267:18199-18204 β1,4 (GnTIV ) EC2.4.1.145 18. human cDNA, Yoshida et al (1998) Glycoconjugate Journal15:1115-1123 19. bovine cDNA, Minowa et al., European Patent EP 0 905232 20. β1,6 (GnT V) EC 2.4.1.155 21. human cDNA, Saito et al (1994)Biochem. Biophys. Res. Commun. 198:318-327 22. rat cDNA, Shoreibah et al(1993) J. Biol. Chem. 268:15381-15385 β1,4 Galactosyltransferase, EC2.4.1.90 (LacNAc synthetase) EC 2.4.1.22 (lactose synthetase) 23. bovinecDNA, D'Agostaro et al (1989) Eur. J. Biochem. 183:211-217 24. bovinecDNA (partial), Narimatsu et al (1986) Proc. Natl. Acad. Sci. U.S.A.83:4720-4724 25. bovine cDNA (partial), Masibay & Qasba (1989) Proc.Natl. Acad. Sci. U.S.A. 86:5733-5377 26. bovine cDNA (5′ end), Russo etal (1990) J. Biol. Chem. 265:3324 27. chicken cDNA (partial), Ghosh etal (1992) Biochem. Biophys. Res. Commun. 1215-1222 28. human cDNA, Masriet al (1988) Biochem. Biophys. Res. Commun. 157:657-663 29. human cDNA,(HeLa cells) Watzele & Berger (1990) Nucl. Acids Res. 18:7174 30. humancDNA, (partial) Uejima et al (1992) Cancer Res. 52:6158-6163 31. humancDNA, (carcinoma) Appert et al (1986) Biochem. Biophys. Res. Commun.139:163-168 32. human gene, Mengle-Gaw et al (1991) Biochem. Biophys.Res. Commun. 176:1269-1276 33. murine cDNA, Nakazawa et al (1988) J.Biochem. 104:165-168 34. murine cDNA, Shaper et al (1988) J. Biol. Chem.263:10420-10428 35. murine cDNA (novel), Uehara & Muramatsu unpublished36. murine gene, Hollis et al (1989) Biochem. Biophys. Res. Commun.162:1069-1075 37. rat protein (partial), Bendiak et al (1993) Eur. J.Biochem. 216:405-417 2,3-Sialyltransferase, (ST3Gal II) (N-linked)(Gal-1,3/4-GlcNAc) EC 2.4.99.6 38. human cDNA, Kitagawa & Paulson (1993)Biochem. Biophys. Res. Commun. 194:375-382 39. rat cDNA, Wen et al(1992) J. Biol. Chem. 267:21011-21019 40. murine cDNA Lee et al., (1994)J. Biol. Chem. 269:10028-10033 2,6-Sialyltransferase, (ST6Gal I) EC2.4.99.1 41. chicken, Kurosawa et al (1994) Eur. J. Biochem 219:375-38142. human cDNA (partial), Lance et al (1989) Biochem. Biophys. Res.Commun. 164:225- 232 43. human cDNA, Grundmann et al (1990) Nucl. AcidsRes. 18:667 44. human cDNA, Zettlmeisl et al (1992) Patent EP0475354-A/345. human cDNA, Stamenkovic et al (1990) J. Exp. Med. 172:641-643 (CD75)46. human cDNA, Bast et al (1992) J. Cell Biol. 116:423-435 47. humangene (partial), Wang et al (1993) J. Biol. Chem. 268:4355-4361 48. humangene (5′ flank), Aasheim et al (1993) Eur. J. Biochem. 213:467-475 49.human gene (promoter), Aas-Eng et al (1995) Biochim. Biophys. Acta1261:166-169 50. mouse cDNA, Hamamoto et al (1993) Bioorg. Med. Chem.1:141-145 51. rat cDNA, Weinstein et al (1987) J. Biol. Chem.262:17735-17743 52. rat cDNA (transcript fragments), Wang et al (1991)Glycobiology 1:25-31, Wang et al (1990) J. Biol. Chem. 265:17849-1785353. rat cDNA (5′ end), O'Hanlon et al (1989) J. Biol. Chem.264:17389-17394; Wang et al (1991) Glycobiology 1:25-31 54. rat gene(promoter), Svensson et al (1990) J. Biol. Chem. 265:20863-20688 55. ratmRNA (fragments), Wen et al (1992) J. Biol. Chem. 267:2512-2518

Additional methods and reagents that can be used in the methods formodifying the glycosylation are described in the literature, such asU.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No.6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat.No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S.Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854,U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No.5,047,335. Appropriate yeast expression systems can be obtained fromsources such as the American Type Culture Collection, Rockville, Md.Vectors are commercially available from a variety of sources.

SEQUENCE LISTINGSSEQ ID NO: 1-6 can be found in U.S. patent application No. 09/892,591SEQ ID NO: 7Primer: regions of high homology within 1,6 mannosyltransferases5′-atggcgaaggcagatggcagt-3′ SEQ ID NO: 8Primer: regions of high homology within 1,6 mannosyltransferases5′-ttagtccttccaacttccttc-3′ SEQ ID NO: 9internal primer: 5′-actgccatctgccttcgccat-3′ SEQ ID NO: 10internal primer: 5′-GTAATACGACTCACTATAGGGC-3′ T7 SEQ ID NO: 11Internal primer: 5′-AATTAACCCTCACTAAAGGG-3′ T3 SEQ ID NO: 12Primer: atgcccgtgg ggggcctgtt gccgctcttc agtagc SEQ ID NO: 13Primer: tcatttctct ttgccatcaa tttccttctt ctgttcacgg SEQ ID NO: 14Primer: ggcgcgccga ctcctccaag ctgctcagcg gggtcctgtt ccac SEQ ID NO: 15Primer: ccttaattaa tcatttctct ttgccatcaa tttccttctt ctgttcacggSEQ ID NO: 16Primer: ggcgagctcg gcctacccgg ccaaggctga gatcatttgt ccagcttcagaSEQ ID NO: 17Primer: gcccacgtcg acggatccgt ttaaacatcg attggagagg ctgacaccgc tactaSEQ ID NO: 18Primer: cgggatccac tagtatttaa atcatatgtg cgagtgtaca actcttccca catggSEQ ID NO: 19Primer: ggacgcgtcg acggcctacc cggccgtacg aggaatttct cggatgactc ttttcSEQ ID NO: 20 Primer: cgggatccct cgagagatct tttttgtaga aatgtcttgg tgcctSEQ ID NO: 21Primer: ggacatgcat gcactagtgc ggccgccacg tgatagttgt tcaattgatt gaaataggga caaSEQ ID NO: 22Primer: ccttgctagc ttaattaacc gcggcacgtc cgacggcggc ccacgggtcc caSEQ ID NO: 23Primer: ggacatgcat gcggatccct taagagccgg cagcttgcaa attaaagcct tcgagcgtcc cSEQ ID NO: 24Primer: gaaccacgtc gacggccatt gcggccaaaa ccttttttcc tattcaaaca caaggcattg cSEQ ID NO: 25 Primer: ctccaatact agtcgaagat tatcttctac ggtgcctgga ctcSEQ ID NO: 26Primer: tggaaggttt aaacaaagct agagtaaaa tagatatagc gagattagag aatgSEQ ID NO: 27Primer: aagaattcgg ctggaaggcc ttgtaccttg atgtagttcc cgttttcatcSEQ ID NO: 28Primer: gcccaagccg gccttaaggg atctcctgat gactgactca ctgataataa aaatacggSEQ ID NO: 29Primer: gggcgcgta tttaaatacta gtggatctat cgaatctaaa tgtaagttaa aatctctaaSEQ ID NO: 30 Primer: ggccgcctgc agatttaaat gaattcgg cgcgccttaatSEQ ID NO: 31 Primer: taaggcgcgc cgaattcatt taaatctgca gggcSEQ ID NO: 32 Primer: 5′-tggcaggcgcgcctcagtcagcgctctcg-3′ SEQ ID NO: 33Primer: 5′-aggttaatta agtgctaattccagctagg-3′ SEQ ID NO: 34primer for K.lactis OCH1 gene: ccagaagaat tcaattytgy cartggSEQ ID NO: 35 primer for K.lactis OCH1 gene: cagtgaaaat acctggnccn gtccaSEQ ID NO: 36primer for K.lactis MNN1 gene: tgccatcttt taggtccagg cccgttcSEQ ID NO: 37primer for K.lactis MNN1 gene: gatcccacga cgcatcgtat ttctttcSEQ ID NO: 38DNA sequence of the 302 bp segment of the putative KlOCH1 gene:gcccttcagtgaaaatacctggcccggtccagttcataatatcggtaccatctgtatttttggcggttttcttttgttgatgtttgtaatttttgttgaacttctttttatccctcatgttgacattataatcatctgcaatgtcttttaatacttcagcatcatctaaaggaatgctgcttttaacatttgccacgctctccaatgttgttgcggtgatatttgtgatcaattcgcgcaataatggatggccagattttgattgtattgtccactgacaaaattgaattctctggaagggc SEQ ID NO: 39Translation of putative KlOCH1 gene (excluding primers):TIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNMRDKKKFNKNYKHQQKKTAKNTDGTDIMN SEQ ID NO: 40DNA sequence of the 405 bp segment of the putative KlMNN1 gene:cccagcgtgccattaccgtatttgccgccgtttgaaatactcaatattcatgatggttgtaaggcgttttttatcattcgcgatataatatgccatcttttaggtccaggcccgttctcttagctatctttggtgtctgtgctaccgtgatatggtacctattctttttccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgatSEQ ID NO: 41 DNA sequence of the K.lactis OCH1 gene:atggggttaccaaagatttcaagaagaacgaggtacattattgtcattgtgctgatactgtacttattgttttctgtgcaatggaatactgcgaaagtgaatcaccatttctataacagcattggcacggtgcttcccagtacagctcgcgtggatcacttgaacttgaaaaacttggacttagcaggtacgagcaataacggtgatcatttgatggatctacgagttcaattggctagtcaattcccctacgattctcgagtacccatccccaaaaaggtatggcagacctggaagattgatcccagttcaaagtcacaggtttcttccatttcaaaatgccagaatgattggaaacatttcagtgcatccgaggaaccgccatatcaataccaattaatcacagatgatcaaatgataccacttctagagcagctatatggtggggtcccacaagtgataaaggcttttgaatccttgccacttccaattcttaaagcagactttttcagatacttgatcctttatgcaagaggtggtatatattctgacatggatacgttcccattaaagccattgtcgtcatggccatcgacttctcagtcctacttttctagtttaaagaatccacaaaggtatagaaattccttggacaaccttgaaacgctagaagcttcagaacctggctttgtcattggtatcgaggctgatccggatagaagcgattgggcagagtggtacgccaggagaatacaattctgtcagtggacaatacaatcaaaatctggccatccattattgcgcgaattgatcacaaatatcaccgcaacaacattggagagcgtggcaaatgttaaaagcagcattcctttagatgatgctgaagtattaaaagacattgcagatgattataatgtcaacatgagggataaaaagaagttcaacaaaaattacaaacatcaacaaaagaaaaccgccaaaaatacagatggtaccgatattatgaactggactggtccaggtattttttcagatgttattttccagtatcttaataacgttatccagaagaatgatgacattttaattttcaatgataatcttaatgttatcaacaaacatggatccaaacatgatacaactatgagattctataaagacattgttaaaaatttacaaaacgacaaaccctcattgttctggggattcttttcattgatgacagagcctattctagtggacgacatcatggtacttccgattacttctttctcaccaggtatcagaacaatgggcgctaaagaagacaacgacgagatggcatttgttaagcatatttttgaaggaagttggaaagactga SEQ ID NO: 42 Translation of putative K.lactis OCH1 gene:MGLPKISRRTRYIIVIVLILYLLFSVQWNTAKVNHHFYNSIGTVLPSTARVDHLNLKNLDLAGTSNNGDHLMDLRVQLASQFPYDSRVPIPKKVWQTWKIDPSSKSQVSSISKCQNDWKHFSASEEPPYQYQLITDDQMIPLLEQLYGGVPQVIKAFESLPLPILKADFFRYLILYARGGIYSDMDTFPLKPLSSWPSTSQSYFSSLKNPQRYRNSLDNLETLEASEPGFVIGIEADPDRSDWAEWYARRIQFCQWTIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNMRDKKKFNKNYKHQQKKTAKNTDGTDIMNWTGPGIFSDVIFQYLNNVIQKNDDILIFNDNLNVINKHGSKHDTTMRFYKDIVKNLQNDKPSLFWGFFSLMTEPILVDDIMVLPITSFSPGIRTMGAKEDNDEMAFVKHIFEGSWKDZ SEQ ID NO: 43DNA sequence of the K.lactis MNN1 gene:atgatggttgtaaggcgttttttatcagcttcgcgatataatatgccatcttttaggtccaggcccgttctcttagctatctttggtgtctgtgctaccgtgatatggtacctattctttttccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgatcttcatacgaaatgtgagttttatttccagaatttatacaatttgaacgaggattggaccaataatattcggacgttcactttcgatattaacgatgtagacacgtctacgaaaattgacgctcttaaagattccgatggggttcaattggtggacgagaaggctatacgtttatacaagagaacgcataacgttgccttggctacggaaaggttacgtctttatgataaatgttttgtcaatagtccaggttcaaacccattgaaaatggatcaccttttcagatcgaacaagaagagtaagactacggctttggatgacgaagtcactgggaaccgtgatacttttaccaagacgaagaaaacttcgttcttaagcgatatggacacgagtagtttccagaagtacgatcaatgggatttcgaacatagaatgttccccatgatcccatatttcgaggaacacaatttcaccaacgtgatgcctattttcaccggctcaaacggtggggaacctttacctcaagggaaattcccggtattagatccaaaatccggtgaattgttacgtgtagagactttcagatatgataaatcgaaatcgctttggaagaactggaatgatatgtcctctgcttctggtaaacgtggtattatcttggctgctggcgacggccaagtggaccaatgcatccgtcttattgctacgttgagagctcaaggaaacgctctacctattcaaattatccacaacaaccaattgaatgagaaatctgtgaaactgttatcggaggccgctaaatctaccgaattctcatccggtagagctcaatctctttggttagtgaatgtgggccccacgttggaatcttcaatgaagagcaattttgggagatttaagaataagtggttgtcagttattttcaacacttttgaagaatttatattcatagatacagatgccatctcctacattaatatggctgattatttcaacttcaaggagtacaaatctactggaacactcttctttaaggataggtctttggcaattggaactgaacagaaatgtggtcctttgttcgaaactcttgaaccaagaattcttgaaatgtactatttcaatactttacctatgatcaatggtgattacgtggaacagcaatgtatgggcatgctcaccccagaggaaaaagtttacaaacgtttctttgaagttggtcatcaacacaacttggaaagtggattattggccatcaacaaaaacgaacacatcatgggattggttactgcaacagtcttaaatatcgcaccaaaggtcggaggttgcggttggggtgacaaagagtttttctggcttggtttgttggttgctggccaacgctactcgatctatgatatagatgcaagtgcaattggtgttcctcaacagaagcaatctatcgctaacggagacgaatttgatgaatataggatttgttctttacaagtggcacatacttcatacgacggacatttactatggataaatggtggctctcagtactgtaagaaaccagagacttttgaaggtgattggaccaacattaaggagcttcgtgaatcgtattctgatgataaagaaaaggctctgaaggcttatagtgatacagttaaggtggaagcagcaatcgtgccagattccagaagtaatggttggggtagagacgatcaaagatgtaaaggctacttctggtgcggcaaatttacttcaaagctgaaaccgtatacttataacacggtggtaactaaaggtgatttgatccgtttcggagacgaggaaatcgaaagtatctccaagattaataagatctggaatgatgctattattccagacggagcttaa SEQ ID NO: 44Translation of putative K.lactis MNN1 gene:MMVVRRFLSASRYNMPSFRSRPVLLAIFGVCATVIWYLFFFQSNLKMADLKKVATSRYLSQEPSLSELMSNVKIKPIEETPVSPLELIPDIEISTRKKYDASWDLLFRGRKYKSFNDYDLHTKCEFYFQNLYNLNEDWTNNIRTFTFDINDVDTSTKIDALKDSDGVQLVDEKAIRLYKRTHNVALATERLRLYDKCFVNSPGSNPLKMDHLFRSNKKSKTTALDDEVTGNRDTFTKTKKTSFLSDMDTSSFQKYDQWDFEHRMFPMIPYFEEHNFTNVMPIFTGSNGGEPLPQGKFPVLDPKSGELLRVETFRYDKSKSLWKNWNDMSSASGKRGIILAAGDGQVDQCIRLIATLRAQGNALPIQIIHNNQLNEKSVKLLSEAAKSTEFSSGRAQSLWLVNVGPTLESSMKSNFGRFKNKWLSVIFNTFEEFIFIDTDAISYINMADYFNFKEYKSTGTLFFKDRSLAIGTEQKCGPLFETLEPRILEMYYFNTLPMINGDYVEQQCMGMLTPEEKVYKRFFEVGHQHNLESGLLAINKNEHIMGLVTATVLNIAPKVGGCGWGDKEFFWLGLLVAGQRYSIYDIDASAIGVPQQKQSIANGDEFDEYRICSLQVAHTSYDGHLLWINGGSQYCKKPETFEGDWTNIKELRESYSDDKEKALKAYSDTVKVEAAIVPDSRSNGWGRDDQRCKGYFWCGKFTSKLKPYTYNTVVTKGDLIRFGDEEIESISKINKIWNDAIIP DGASEQ ID NO: 45-56: See Table 12. Primer name Primer sequence NeuA sense5′- ATGAGAACAAAAATTATTGCGATAATTCCAGCCCG- 3′ (SEQ ID NO: 45)NeuA antisense 5′-TCATTTAACAATCTCCGCTATTTCGTTTTC-3′ (SEQ ID NO: 46)NeuB sense 5′- ATGAGTAATATATATATCGTTGCTGAAATTGGTTG- 3′ (SEQ ID NO: 47)NeuB antisense 5′-TTATTCCCCCTGATTTTTGAATTCGCTATG-3′ (SEQ ID NO: 48)NeuC sense 5′- ATGAAAAAAATATTATACGTAACTGGATCTAGAG- 3′ (SEQ ID NO: 49)NeuC antisense 5′-CTAGTCATAACTGGTGGTACATTCCGGGATGTC- 3′ (SEQ ID NO: 50)mouse CMP-Sia 5′-ATGGACGCGCTGGAGAAGGGGGCCGTCACGTC- synthase sense 3′(SEQ ID NO: 51) mouse CMP-Sia 5′- synthase antisenseCTATTTTTGGCATGAGTTATTAACTTTTTCTATCAG- 3′ (SEQ ID NO: 52) porcine GlcNAc5′-ATGGAGAAGGAGCGCGAAACTCTGCAGG-3′ epimerase sense (SEQ ID NO: 53)porcine GlcNAc 5′-CTAGGCGAGGCGGCTCAGCAGGGCGCTC-3′ epimerase(SEQ ID NO: 54) antisense E. coli Sialate5′-ATGGCAACGAATTTACGTGGCGTAATGGCTG-3′ aldolase sense (SEQ ID NO: 55)E. coli Sialate 5′-TCACCCGCGCTCTTGCATCAACTGCTGGGC-3′ aldolase antisense(SEQ ID NO: 56)SEQ ID NO: 57-68: Are disclosed throughout the specification.SEQ ID NO: 69-76: See Table 12. mouse bifunctional 5′- UDP-N-ATGGAGAAGAACGGGAACAACCGAAAGCTCCG-3′ acetylglucosamine- (SEQ ID NO: 69)2-epimerase/N- acetylmannosamine kinase sense mouse bifunctional5′-CTAGTGGATCCTGCGCGTTGTGTAGTCCAG-3′ UDP-N- (SEQ ID NO: 70)acetylglucosamine- 2-epimerase/N- acetylmannosamine kinase antisensemouse Sia9P syn 5′-ATGCCGCTGGAACTGGAGCTGTGTCCCGGGC-3′ sense(SEQ ID NO: 71) mouse Sia9P syn 5′-TTAAGCCTTGATTTTCTTGCTGTGACTTTCCAC-antisense 3′ (SEQ ID NO: 72) human N- 5′- acetylneuraminicGGGAGAATGCGGCCGCCACCATGGGGCTGAGCCG acid phosphataseC GTGCGGGCG GTTTTC-3′ (SEQ ID NO: 73) (NANP) sense human N- 5′-acetylneuraminic GTATAGACTGCAAAGTCAGTATGTCCACTTGATT acid phosphataseAATTAACC-3′ (SEQ ID NO: 74) (NANP) antisense human CMP-Sia5′-ATGGACTCGGTGGAGAAGGGGGCCGCCACC-3′ synthase sense (SEQ ID NO: 75)human CMP-Sia 5′-CTATTTTTGGCATGAATTATTAACTTTTTCC-3′ synthase antisense(SEQ ID NO: 76) SEQ ID NO: 77 Primer for hGNE:GGGAGAATGCGGCCGCCACCATGGAGAAGAATGGAAATAACCGAAAGCT GCG SEQ ID NO: 78Primer for hGNE: CCTTAATTAACTAGTAGATCCTGCGTGTTGTGTAGTCCAGAACSEQ ID NO: 79 Primer for hSiaPsynGGGAGAATGCGGCCGCCACCATGCCGCTGGAGCTGGAGCTGTGTCCCG SEQ ID NO: 80Primer for hSiaPsyn CCTTAATTAATTAAGACTTGATTTTTTTGCCATGATTATCTACCSEQ ID NO: 81 Primer GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTCSEQ ID NO: 82 Primer CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAGSEQ ID NO: 83 Primer GGCTCGAGCGGCCGCCACCATGAATAGCATACACATGAACGCCAATACGSEQ ID NO: 84 Primer CCCTCGAGTTAATTAACTAGACGCGCGGCAGCAGCTTCTCCTCATCG-3′)SEQ ID NO: 85 Primer ATG ACT GGT GTT CAT GAA GGG SEQ ID NO: 86 PrimerTTA CTT ATA TGT CTT GGT ATG SEQ ID NO: 87 PrimerGCG GCC GCA TGA CTG GTG TTC ATG AAG GGA CTG TGT TGG TTA CTGGCG GCG CTG GTT ATA TAG GTT CTC ATA CGT GCG TTG TTT TGT TAG AAA ASEQ ID NO: 88 Primer TTA ATT AAT TAC TTA TAT GTC TTG GTA TG 3′)SEQ ID NO: 89 Primer GCCGCGACCTGAGCCGCCTGCCCCAAC SEQ ID NO: 90 PrimerCTAGCTCGGTGTCCCGATGTCCACTGT SEQ ID NO: 91 PrimerCTTAGGCGCGCCGGCCGCGACCTGAGCCGCCTGCCC SEQ ID NO: 92 PrimerGGGGCATATCTGCCGCCCATC SEQ ID NO: 93 Primer GATGGGCGGCAGATATGCCCCSEQ ID NO: 94 Primer CTTCTTAATTAACTAGCTCGGTGTCCCGATGTCCAC SEQ ID NO: 95Primer GGGAGAATGCGGCCGCCACCATGGACTCGGTGGAGAAGGGGGCCGCCAC CTCSEQ ID NO: 96 Primer CCTTAATTAACTATTTTTGGCATGAATTATTAACTTTTTCCATTASEQ ID NO: 97 Primer CGGAATTCCACCATGGCTCCGGCGAGAGAAAATGTCAGSEQ ID NO: 98 Primer CGGAATTCTCACACACCAATGATTCTCTCTTTTGAAG SEQ ID NO: 99Primer GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC SEQ ID NO: 100 PrimerCCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG SEQ ID NO: 101 PrimerGGCGCGCCAGCAAGCAAGACCCTAAGGAAGACATTCC SEQ ID NO: 102 PrimerCCTTAATTAATCAACAACGAATGTTCCGGAAGCCAGAAAGG SEQ ID NO: 103 and 104Nucleic acid (SEQ ID NO: 103) and amino acid sequence (SEQ ID NO: 104) of thecodon - optimized hST6Gal leader 53 fusion (FIG. 33)

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1. A method for producing a recombinant sialylated glycoprotein in aPichia pastoris host cell, the host cell selected or engineered toproduce glycoproteins comprising a Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂glycoform, the method comprising the step of transforming into the hostcell: a. a nucleic acid encoding an enzyme having sialyltransferaseactivity, wherein the nucleic acid encodes a silayltransferase enzymecomprising the amino acid sequence of SEQ ID NO:104 or a catalyticdomain thereof; b. a nucleic acid encoding a CMP-sialic acidtransporter; and c. one or more nucleic acids encoding a CMP-sialic acidpathway consisting of: a bifunctionalUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, anN-acetylneuraminate-9-phosphate synthase, and a CMP-sialic acidsynthase; wherein, upon passage of the recombinant glycoprotein throughthe secretory pathway of the host cell, a recombinant glycoproteincomprising a NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform isproduced. 2-11. (canceled)
 12. The method of claim 1, wherein said hostcell comprises a cellular pool of CMP-sialic acid. 13-15. (canceled) 16.The method of claim 1, wherein the method further comprises culturingsaid host cell in the presence of a sialic acid donor or a precursor ofa sialic acid donor.
 17. The method of claim 1, wherein the methodfurther comprises the step of introducing into the host cell one or moreadditional nucleic acids encoding one or more enzymes selected from thegroup consisting of glycosyltransferases, glycosidases and sugartransporters. 18-34. (canceled)
 35. The method of claim 1, wherein thehost cell is transformed with: a. a nucleic acid encoding a mouseCMP-sialic acid transporter; and b. one or more nucleic acids encodinga: human a bifunctionalUDP-N_acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, a humanN-acetylneuraminate-9-phosphate synthase, and a human CMP-sialic acidsynthase;